Humanized fc gamma riib-specific antibodies and methods of use thereof

ABSTRACT

The present invention relates to humanized FcγRIIB antibodies, fragments, and variants thereof that bind human FcγRIIB with a greater affinity than said antibody binds FcγRIIA. The invention encompasses the use of the humanized antibodies of the invention for the treatment of any disease related to loss of balance of Fc receptor mediated signaling, such as cancer, autoimmune and inflammatory disease. The invention provides methods of enhancing the therapeutic effect of therapeutic antibodies by administering the humanized antibodies of the invention to enhance the effector function of the therapeutic antibodies. The invention also provides methods of enhancing the efficacy of a vaccine composition by administering the humanized antibodies of the invention. The invention encompasses methods for treating an autoimmune disease and methods for elimination of cancer cells that express FcγRIIB.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/785,117, filed May 21, 2010, which is a divisional of U.S. patentapplication Ser. No. 11/754,015, filed May 25, 2007 (issued Aug. 31,2010 as U.S. Pat. No. 7,786,270), which application claims benefit ofU.S. Provisional Patent Application Ser. No. 60/809,116, filed May 26,2006 (lapsed), and U.S. Provisional Patent Application No. 60/816,126,filed Jun. 23, 2006 (lapsed), which applications are herein incorporatedby reference in their entireties and to which priority is claimed.

2. FIELD OF THE INVENTION

The present invention relates to humanized FcγRIIB antibodies,fragments, and variants thereof that bind human FcγRIIB with a greateraffinity than said antibody binds FcγRIIA. The invention encompasses theuse of the humanized antibodies of the invention for the treatment ofany disease related to loss of balance of Fc receptor mediatedsignaling, such cancer (preferably a B-cell malignancy, particularly,B-cell chronic lymphocytic leukemia or non-Hodgkin's lymphoma),autoimmune disease, inflammatory disease or IgE-mediated allergicdisorder. The present invention also encompasses the use of a humanizedFcγRIIB antibody or an antigen-binding fragment thereof, in combinationwith other cancer therapies. The invention provides methods of enhancingthe therapeutic effect of therapeutic antibodies by administering thehumanized antibodies of the invention to enhance the effector functionof the therapeutic antibodies. The invention also provides methods ofenhancing the efficacy of a vaccine composition by administering thehumanized antibodies of the invention with a vaccine composition.

3. BACKGROUND OF THE INVENTION

3.1 Fc Receptors and their Roles in the Immune System

The interaction of antibody-antigen complexes with cells of the immunesystem results in a wide array of responses, ranging from effectorfunctions such as antibody-dependent cytotoxicity, mast celldegranulation, and phagocytosis to immunomodulatory signals such asregulating lymphocyte proliferation and antibody secretion. All theseinteractions are initiated through the binding of the Fc domain ofantibodies or immune complexes to specialized cell surface receptors onhematopoietic cells. The diversity of cellular responses triggered byantibodies and immune complexes results from the structuralheterogeneity of Fc receptors. Fc receptors share structurally relatedligand binding domains which presumably mediate intracellular signaling.

The Fc receptors, members of the immunoglobulin gene superfamily ofproteins, are surface glycoproteins that can bind the Fc portion ofimmunoglobulin molecules. Each member of the family recognizesimmunoglobulins of one or more isotypes through a recognition domain onthe α chain of the Fc receptor. Fc receptors are defined by theirspecificity for immunoglobulin subtypes. Fc receptors for IgG arereferred to as FcγR, for IgE as FcεR, and for IgA as FcαR. Differentaccessory cells bear Fc receptors for antibodies of different isotype,and the isotype of the antibody determines which accessory cells will beengaged in a given response (reviewed by Ravetch J. V. et al. 1991,Annu. Rev. Immunol. 9: 457-92; Gerber J. S. et al. 2001 Microbes andInfection, 3: 131-139; Billadeau D. D. et al. 2002, The Journal ofClinical Investigation, 2(109): 161-168; Ravetch J. V. et al. 2000,Science, 290: 84-89; Ravetch J. V. et al., 2001 Annu. Rev. Immunol.19:275-90; Ravetch J. V. 1994, Cell, 78(4): 553-60; each of which isincorporated by reference herein in its entirety). The different Fcreceptors, the cells that express them, and their isotype specificity issummarized in Table 1 (adapted from Immunobiology: The Immune System inHealth and Disease, 4^(th) ed. 1999, Elsevier Science Ltd/GarlandPublishing, New York, incorporated herein by reference in its entirety).

Fcγ Receptors

Each member of this family is an integral membrane glycoprotein,possessing extracellular domains related to a C2-set ofimmunoglobulin-related domains, a single membrane spanning domain and anintracytoplasmic domain of variable length. There are three known FcγRs,designated FcγRI(CD64), FcγRII(CD32), and FcγRIII(CD16). The threereceptors are encoded by distinct genes; however, the extensive homologyamong the three family members suggest they arose from a commonprogenitor perhaps by gene duplication. This invention specificallyfocuses on FcγRII(CD32).

FcγRII(CD32)

FcγRII proteins are 40 KDa integral membrane glycoproteins which bindonly the complexed IgG due to a low affinity for monomeric Ig (10⁶ M⁻¹).This receptor is the most widely expressed FcγR, present on allhematopoietic cells, including monocytes, macrophages, B cells, NKcells, neutrophils, mast cells, and platelets. FcγRII has only twoimmunoglobulin-like regions in its immunoglobulin binding chain andhence a much lower affinity for IgG than FcγRI. There are three humanFcγRII genes (FcγRII-A, FcγRII-B, FcγRII-C), all of which bind IgG inaggregates or immune complexes.

Distinct differences within the cytoplasmic domains of FcγRII-A (CD32A)and FcγRII-B (CD32B) create two functionally heterogeneous responses toreceptor ligation. The fundamental difference is that the A isoforminitiates intracellular signaling leading to cell activation such asphagocytosis and respiratory burst, whereas the β isoform initiatesinhibitory signals, e.g., inhibiting B-cell activation.

Signaling Through FcγRs

Both activating and inhibitory signals are transduced through the FcγRsfollowing ligation. These diametrically opposing functions result fromstructural differences among the different receptor isoforms. Twodistinct domains within the cytoplasmic signaling domains of thereceptor called immunoreceptor tyrosine based activation motifs (ITAMs)or immunoreceptor tyrosine based inhibitory motifs (ITIMS) account forthe different responses. The recruitment of different cytoplasmicenzymes to these structures dictates the outcome of the FcγR-mediatedcellular responses. ITAM-containing FcγR complexes include FcγRI,FcγRIIA, FcγRIIIA, whereas ITIM-containing complexes only includeFcγRIIB.

Human neutrophils express the FcγRIIA gene. FcγRIIA clustering viaimmune complexes or specific antibody cross-linking serves to aggregateITAMs along with receptor-associated kinases which facilitate ITAMphosphorylation. ITAM phosphorylation serves as a docking site for Sykkinase, activation of which results in activation of downstreamsubstrates (e.g., PI₃K). Cellular activation leads to release ofproinflammatory mediators.

The FcγRIIB gene is expressed on B lymphocytes; its extracellular domainis 96% identical to FcγRIIA and binds IgG complexes in anindistinguishable manner. The presence of an ITIM in the cytoplasmicdomain of FcγRIIB defines this inhibitory subclass of FcγR. Recently themolecular basis of this inhibition was established. When co-ligatedalong with an activating FcγR, the ITIM in FcγRIIB becomesphosphorylated and attracts the SH2 domain of the inositol polyphosphate5′-phosphatase (SHIP), which hydrolyzes phosphoinositol messengersreleased as a consequence of ITAM-containing FcγR-mediated tyrosinekinase activation, consequently preventing the influx of intracellularCa⁺⁺. Thus, crosslinking of FcγRIIB dampens the activating response toFcγR ligation and inhibits cellular responsiveness. B cell activation, Bcell proliferation and antibody secretion is thus aborted.

TABLE 1 Receptors for the Fc Regions of Immunoglobulin Isotypes FcγRIFcγRII-A FcγRII-B2 FcγRII-BI FcγRIII FcαRI Receptor (CD64) (CD32) (CD32)(CD32) (CD16) FcεRI (CD89) Binding IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1,10⁸ M⁻¹ 2 × 10⁶ M⁻¹ 2 × 10⁶ M⁻¹ 2 × 10⁶ M⁻¹ 5 × 10⁵ M⁻¹ 10¹⁰ M⁻¹ IgA210⁷ M⁻¹ Cell Type Macrophages Macrophages Macrophages B cells NK cellsMast cells Macrophages Neutrophils Neutrophils Neutrophils Mast cellsEosinophil Eosinophil Neutropils Eosinophils Eosinophils Eosinophilsmacrophages Basophils Eosinophils Dendritic cells Dendritic cellsNeutrophils Platelets Mast Cells Langerhan cells Effect of Uptake UptakeUptake No uptake Induction of Secretion of Uptake Ligation StimulationGranule Inhibition of Inhibition of Killing granules InductionActivation of release Stimulation Stimulation of killing respiratoryburst Induction of killing

3.2 Diseases of Relevance

3.2.1 Cancer

A neoplasm, or tumor, is a neoplastic mass resulting from abnormaluncontrolled cell growth which can be benign or malignant. Benign tumorsgenerally remain localized. Malignant tumors are collectively termedcancers. The term “malignant” generally means that the tumor can invadeand destroy neighboring body structures and spread to distant sites tocause death (for review, see Robbins and Angell, 1976, Basic Pathology,2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-122, incorporatedherein by reference in its entirety). Cancer can arise in many sites ofthe body and behave differently depending upon its origin. Cancerouscells destroy the part of the body in which they originate and thenspread to other part(s) of the body where they start new growth andcause more destruction.

More than 1.2 million Americans develop cancer each year. Cancer is thesecond leading case of death in the United States and if current trendscontinue, cancer is expected to be the leading cause of the death by theyear 2010. Lung and prostate cancer are the top cancer killers for menin the United States. Lung and breast cancer are the top cancer killersfor women in the United States. One in two men in the United States willbe diagnosed with cancer at some time during his lifetime. One in threewomen in the United States will be diagnosed with cancer at some timeduring her lifetime.

A cure for cancer has yet to be found. Current treatment options, suchas surgery, chemotherapy and radiation treatment, are oftentimes eitherineffective or present serious side effects.

3.2.1.1 B-Cell Malignancies

B cell malignancies, including, but not limited to, B-cell lymphomas andleukemias, are neoplastic diseases with significant incidence in theUnited States. There are approximately 55,000 new lymphoma cases of peryear in the U.S. (1998 data), with an estimated 25,000 deaths per year.This represents 4% of cancer incidence and 4% of all cancer-relateddeaths in the U.S. population. The revised European-Americanclassification of lymphoid neoplasms (1994 REAL classification, modified1999; incorporated herein by reference in its entirety) groupedlymphomas based on their origin as either B cell lineage lymphoma, Tcell lineage lymphoma, or Hodgkin's lymphoma. Lymphoma of the B celllineage is the most common type of non-Hodgkin's lymphoma (NHL)diagnosed in the U.S. (Williams, Hematology 6th ed. (Beutler et al.Ed.), McGraw Hill 2001; incorporated herein by reference in itsentirety).

Chronic lymphocytic leukemia (CLL) is a neoplastic disease characterizedby the accumulation of small, mature-appearing lymphocytes in the blood,marrow, and lymphoid tissues. CLL has an incidence of 2.7 cases per100,000 in the U.S. The risk increases progressively with age,particularly in men. It accounts for 0.8% of all cancers and is the mostcommon adult leukemia, responsible for 30% of all leukemias. In nearlyall cases (>98%) the diseased cells belong to the B lymphocyte lineage.A non-leukemic variant, small lymphocytic lymphoma, constitutes 5-10% ofall lymphomas, has histological, morphological and immunologicalfeatures indistinguishable from that of involved lymph nodes in patientswith B-CLL (Williams, 2001).

The natural history of chronic lymphocytic leukemia falls into severalphases. In the early phase, chronic lymphocytic leukemia is an indolentdisease, characterized by the accumulation of small, mature,functionally-incompetent malignant B-cells having a lengthened lifespan. Eventually, the doubling time of the malignant B-cells decreasesand patients become increasingly symptomatic. While treatment withchemotherapeutic agents can provide symptomatic relief, the overallsurvival of the patients is only minimally extended. The late stages ofchronic lymphocytic leukemia are characterized by significant anemiaand/or thrombocytopenia. At this point, the median survival is less thantwo years (Foon et al., 1990, Annals Int. Medicine 113:525; incorporatedherein by reference in its entirety). Due to the very low rate ofcellular proliferation, chronic lymphocytic leukemia is resistant totreatment with chemotherapeutic agents.

Recently, gene expression studies have identified several genes that maybe up regulated in lymphoproliferative disorders. One molecule thoughtto be over-expressed in patients with B-cell chronic lymphocyticleukemia (B-CLL) and in a large fraction of non-Hodgkin lymphomapatients is CD32B (Alizadeh et al., 2000, Nature 403:503-511; Rosenwaldet al., 2001, J. Exp. Med. 184:1639-1647; each of which is incorporatedherein by reference in its entirety). However, the role of CD32B isB-CLL is unclear since one report demonstrates that CD32B was expressedon a low percentage of B-CLL cells and at a low density (Damle et al.,2002, Blood 99:4087-4093; incorporated herein by reference in itsentirety). CD32B is a B cell lineage surface antigen, whoseover-expression in B cell neoplasia makes it a suitable target fortherapeutic antibodies. In addition, CD32B belongs to the category ofinhibitory receptors, whose ligation delivers a negative signal.Therefore, antibodies directed against CD32B could function to eliminatetumor cells by mechanisms that include complement dependent cytotoxicity(CDC), antibody-dependent cellular cytotoxicity (ADCC), but alsotriggering an apoptotic signal. The high homology of CD32B with itscounterpart, CD32A, an activating Fc receptor, has thus far hampered thegeneration of antibodies that selectively recognize one but not theother form of the molecule.

3.2.1.2 Cancer Therapy

Currently, cancer therapy may involve surgery, chemotherapy, hormonaltherapy and/or radiation treatment to eradicate neoplastic cells in apatient (See, for example, Stockdale, 1998, “Principles of CancerPatient Management”, in Scientific American: Medicine, vol. 3,Rubenstein and Federman, eds., Chapter 12, Section IV; each of which isincorporated herein by reference in its entirety). Cancer therapy canalso involve biological therapy or immunotherapy. All of theseapproaches pose significant drawbacks for the patient. Surgery, forexample, may be contraindicated due to the health of the patient or maybe unacceptable to the patient. Additionally, surgery may not completelyremove the neoplastic tissue. Radiation therapy is only effective whenthe neoplastic tissue exhibits a higher sensitivity to radiation thannormal tissue, and radiation therapy can also often elicit serious sideeffects. Hormonal therapy is rarely given as a single agent and,although it can be effective alone, is often used to prevent or delayrecurrence of cancer after other treatments have removed the majority ofthe cancer cells. Biological therapies/immunotherapies are limited innumber and may produce side effects such as rashes or swellings,flu-like symptoms, including fever, chills and fatigue, digestive tractproblems or allergic reactions.

With respect to chemotherapy, there are a variety of chemotherapeuticagents available for treatment of cancer. A significant majority ofcancer chemotherapeutics act by inhibiting DNA synthesis, eitherdirectly, or indirectly by inhibiting the biosynthesis of thedeoxyribonucleotide triphosphate precursors, to prevent DNA replicationand concomitant cell division (See, for example, Gilman et al., Goodmanand Gilman's: The Pharmacological Basis of Therapeutics, Eighth Ed.(Pergamom Press, New York, 1990); incorporated herein by reference inits entirety). These agents, which include alkylating agents, such asnitrosourea, anti-metabolites, such as methotrexate and hydroxyurea, andother agents, such as etoposides, campathecins, bleomycin, doxorubicin,daunorubicin, etc., although not necessarily cell cycle specific, killcells during S phase because of their effect on DNA replication. Otheragents, specifically colchicine and the vinca alkaloids, such asvinblastine and vincristine, interfere with microtubule assemblyresulting in mitotic arrest. Chemotherapy protocols generally involveadministration of a combination of chemotherapeutic agents to increasethe efficacy of treatment.

Despite the availability of a variety of chemotherapeutic agents,chemotherapy has many drawbacks (See, for example, Stockdale, 1998,“Principles Of Cancer Patient Management” in Scientific AmericanMedicine, vol. 3, Rubenstein and Federman, eds., ch. 12, sect. 10;incorporated herein by reference in its entirety). Almost allchemotherapeutic agents are toxic, and chemotherapy causes significant,and often dangerous, side effects, including severe nausea, bone marrowdepression, immunosuppression, etc. Additionally, even withadministration of combinations of chemotherapeutic agents, many tumorcells are resistant or develop resistance to the chemotherapeuticagents. In fact, those cells resistant to the particularchemotherapeutic agents used in the treatment protocol often prove to beresistant to other drugs, even those agents that act by mechanismsdifferent from the mechanisms of action of the drugs used in thespecific treatment; this phenomenon is termed pleiotropic drug ormultidrug resistance. Thus, because of drug resistance, many cancersprove refractory to standard chemotherapeutic treatment protocols.

B cell malignancy is generally treated with single agent chemotherapy,combination chemotherapy and/or radiation therapy. These treatments canreduce morbidity and/or improve survival, albeit they carry significantside effects. The response of B-cell malignancies to various forms oftreatment is mixed. For example, in cases in which adequate clinicalstaging of non-Hodgkin's lymphoma is possible, field radiation therapycan provide satisfactory treatment. Certain patients, however, fail torespond and disease recurrence with resistance to treatment ensues withtime, particularly with the most aggressive variants of the disease.About one-half of the patients die from the disease (Devesa et al.,1987, J. Nat'l Cancer Inst. 79:701; incorporated herein by reference inits entirety).

Investigational therapies for the treatment of refractory B cellneoplasia include autologous and allogeneic bone marrow or stem celltransplantation and gene therapies. Recently, immunotherapy usingmonoclonal antibodies to target B-cell specific antigens has beenintroduced in the treatment of B cell neoplasia. The use of monoclonalantibodies to direct radionuclides, toxins, or other therapeutic agentsoffers the possibility that such agents can be delivered selectively totumor sites, thus limiting toxicity to normal tissues.

There is a significant need for alternative cancer treatments,particularly for treatment of cancer that has proved refractory tostandard cancer treatments, such as surgery, radiation therapy,chemotherapy, and hormonal therapy. A promising alternative isimmunotherapy, in which cancer cells are specifically targeted by cancerantigen-specific antibodies. Major efforts have been directed atharnessing the specificity of the immune response, for example,hybridoma technology has enabled the development of tumor selectivemonoclonal antibodies (See Green M. C. et al., 2000 Cancer Treat Rev.,26: 269-286; Weiner L M, 1999 Semin Oncol. 26(suppl. 14):43-51; each ofwhich is incorporated herein by reference in its entirety), and in thepast few years, the Food and Drug Administration has approved the firstMAbs for cancer therapy: Rituxin (anti-CD20) for non-Hodgkin's Lymphoma,Campath (anti-CD52) for B-cell chronic lymphocytic leukemia (B-CLL) andHerceptin [anti-(c-erb-2/HER-2)] for metastatic breast cancer (SuzanneA. Eccles, 2001, Breast Cancer Res. 3: 86-90; incorporated herein byreference in its entirety). However, the potency of antibody effectorfunction, e.g., to mediate antibody dependent cellular cytotoxicity(“ADCC”) is an obstacle to such treatment. Furthermore, with Rituxan andCampath, at least half the patients fail to respond and a fraction ofresponders may be refractory to subsequent treatments. Methods toimprove the efficacy of such treatment are thus needed.

There is a need for alternative therapies for cancer, particularly,B-cell malignancies, especially for patients that are refractory forstandard cancer treatments and new immunotherapies such as Rituxan.

3.2.2 Inflammatory Diseases and Autoimmune Diseases

Inflammation is a process by which the body's white blood cells andchemicals protect our bodies from infection by foreign substances, suchas bacteria and viruses. It is usually characterized by pain, swelling,warmth and redness of the affected area. Chemicals known as cytokinesand prostaglandins control this process, and are released in an orderedand self-limiting cascade into the blood or affected tissues. Thisrelease of chemicals increases the blood flow to the area of injury orinfection, and may result in the redness and warmth. Some of thechemicals cause a leak of fluid into the tissues, resulting in swelling.This protective process may stimulate nerves and cause pain. Thesechanges, when occurring for a limited period in the relevant area, workto the benefit of the body.

In autoimmune and/or inflammatory disorders, the immune system triggersan inflammatory response when there are no foreign substances to fightand the body's normally protective immune system causes damage to itsown tissues by mistakenly attacking itself. There are many differentautoimmune disorders that affect the body in different ways. Forexample, the brain is affected in individuals with multiple sclerosis,the gut is affected in individuals with Crohn's disease, and thesynovium, bone and cartilage of various joints are affected inindividuals with rheumatoid arthritis. As autoimmune disorders progress,destruction of one or more types of body tissues, abnormal growth of anorgan, or changes in organ function may result. The autoimmune disordermay affect only one organ or tissue type or may affect multiple organsand tissues. Organs and tissues commonly affected by autoimmunedisorders include red blood cells, blood vessels, connective tissues,endocrine glands (e.g., the thyroid or pancreas), muscles, joints, andskin. Examples of autoimmune disorders include, but are not limited to,Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type 1diabetes, rheumatoid arthritis, systemic lupus erythematosus,dermatomyositis, Sjogren's syndrome, multiple sclerosis, autoimmuneinner ear disease, inflammatory bowel disease, arthritis, myastheniagravis, Reiter's syndrome, Graves disease, autoimmune hepatitis,familial adenomatous polyposis and ulcerative colitis.

Rheumatoid arthritis (RA) and juvenile rheumatoid arthritis are types ofinflammatory arthritis. Arthritis is a general term that describesinflammation in joints. Some, but not all, types of arthritis are theresult of misdirected inflammation. Besides rheumatoid arthritis, othertypes of arthritis associated with inflammation include the following:psoriatic arthritis, Reiter's syndrome, ankylosing spondylitisarthritis, and gouty arthritis. Rheumatoid arthritis is a type ofchronic arthritis that occurs in joints on both sides of the body (suchas both hands, wrists or knees). This symmetry helps distinguishrheumatoid arthritis from other types of arthritis. In addition toaffecting the joints, rheumatoid arthritis may occasionally affect theskin, eyes, lungs, heart, blood or nerves.

Rheumatoid arthritis affects about 1% of the world's population and ispotentially disabling. There are approximately 2.9 million incidences ofrheumatoid arthritis in the United States. Two to three times more womenare affected than men. The typical age that rheumatoid arthritis occursis between 25 and 50. Juvenile rheumatoid arthritis affects 71,000 youngAmericans (aged eighteen and under), affecting six times as many girlsas boys.

Rheumatoid arthritis is an autoimmune disorder where the body's immunesystem improperly identifies the synovial membranes that secrete thelubricating fluid in the joints as foreign. Inflammation results, andthe cartilage and tissues in and around the joints are damaged ordestroyed. In severe cases, this inflammation extends to other jointtissues and surrounding cartilage, where it may erode or destroy boneand cartilage and lead to joint deformities. The body replaces damagedtissue with scar tissue, causing the normal spaces within the joints tobecome narrow and the bones to fuse together. Rheumatoid arthritiscreates stiffness, swelling, fatigue, anemia, weight loss, fever, andoften, crippling pain. Some common symptoms of rheumatoid arthritisinclude joint stiffness upon awakening that lasts an hour or longer;swelling in a specific finger or wrist joints; swelling in the softtissue around the joints; and swelling on both sides of the joint.Swelling can occur with or without pain, and can worsen progressively orremain the same for years before progressing.

The diagnosis of rheumatoid arthritis is based on a combination offactors, including: the specific location and symmetry of painfuljoints, the presence of joint stiffness in the morning, the presence ofbumps and nodules under the skin (rheumatoid nodules), results of X-raytests that suggest rheumatoid arthritis, and/or positive results of ablood test called the rheumatoid factor. Many, but not all, people withrheumatoid arthritis have the rheumatoid-factor antibody in their blood.The rheumatoid factor may be present in people who do not haverheumatoid arthritis. Other diseases can also cause the rheumatoidfactor to be produced in the blood. That is why the diagnosis ofrheumatoid arthritis is based on a combination of several factors andnot just the presence of the rheumatoid factor in the blood.

The typical course of the disease is one of persistent but fluctuatingjoint symptoms, and after about 10 years, 90% of sufferers will showstructural damage to bone and cartilage. A small percentage will have ashort illness that clears up completely, and another small percentagewill have very severe disease with many joint deformities, andoccasionally other manifestations of the disease. The inflammatoryprocess causes erosion or destruction of bone and cartilage in thejoints. In rheumatoid arthritis, there is an autoimmune cycle ofpersistent antigen presentation, T-cell stimulation, cytokine secretion,synovial cell activation, and joint destruction. The disease has a majorimpact on both the individual and society, causing significant pain,impaired function and disability, as well as costing millions of dollarsin healthcare expenses and lost wages (see, for example, the NIH websiteand the NIAID website).

Currently available therapy for arthritis focuses on reducinginflammation of the joints with anti-inflammatory or immunosuppressivemedications. The first line of treatment of any arthritis is usuallyanti-inflammatories, such as aspirin, ibuprofen and Cox-2 inhibitorssuch as celecoxib and rofecoxib. “Second line drugs” include gold,methotrexate and steroids. Although these are well-establishedtreatments for arthritis, very few patients remit on these lines oftreatment alone. Recent advances in the understanding of thepathogenesis of rheumatoid arthritis have led to the use of methotrexatein combination with antibodies to cytokines or recombinant solublereceptors. For example, recombinant soluble receptors and monoclonalantibodies for tumor necrosis factor (TNF)-α have been used incombination with methotrexate in the treatment of arthritis. However,only about 50% of the patients treated with a combination ofmethotrexate and anti-TNF-α agents such as recombinant soluble receptorsfor TNF-α show clinically significant improvement. Many patients remainrefractory despite treatment. Difficult treatment issues still remainfor patients with rheumatoid arthritis. Many current treatments have ahigh incidence of side effects or cannot completely prevent diseaseprogression. So far, no treatment is ideal, and there is no cure. Noveltherapeutics are needed that more effectively treat rheumatoid arthritisand other autoimmune disorders.

3.2.3 Allergy

Immune-mediated allergic (hypersensitivity) reactions are classifiedinto four types (I-IV) according to the underlying mechanisms leading tothe expression of the allergic symptoms. Type I allergic reactions arecharacterized by IgE-mediated release of vasoactive substances such ashistamine from mast cells and basophils. The release of these substancesand the subsequent manifestation of allergic symptoms are initiated bythe cross-linking of allergen-bound IgE to its receptor on the surfaceof mast cells and basophils. In individuals suffering from type Iallergic reactions, exposure to an allergen for a second time leads tothe production of high levels of IgE antibodies specific for theallergen as a result of the involvement of memory B and T cells in the3-cell interaction required for IgE production. The high levels of IgEantibodies produced cause an increase in the cross-linking of IgEreceptors on mast cells and basophils by allergen-bound IgE, which inturn leads to the activation of these cells and the release of thepharmacological mediators that are responsible for the clinicalmanifestations of type I allergic diseases.

Two receptors with differing affinities for IgE have been identified andcharacterized. The high affinity receptor (FcεRI) is expressed on thesurface of mast cells and basophils. The low affinity receptor(FcεRII/CD23) is expressed on many cell types including B cells, Tcells, macrophages, eosinophils and Langerhan cells. The high affinityIgE receptor consists of three subunits (alpha, beta and gamma chains).Several studies demonstrate that only the alpha chain is involved in thebinding of IgE, whereas the beta and gamma chains (which are eithertransmembrane or cytoplasmic proteins) are required for signaltransduction events. The identification of IgE structures required forIgE to bind to the FcεRI on mast cells and basophils is of utmostimportance in devising strategies for treatment or prevention ofIgE-mediated allergies. For example, the elucidation of the IgEreceptor-binding site could lead to the identification of peptides orsmall molecules that block the binding of IgE to receptor-bearing cellsin vivo.

Currently, IgE-mediated allergic reactions are treated with drugs suchas antihistamines and corticosteroids which attempt to alleviate thesymptoms associated with allergic reactions by counteracting the effectsof the vasoactive substances released from mast cells and basophils.High doses of antihistamines and corticosteroids have deleterious sideeffects (e.g., central nervous system disturbance, constipation, etc).Thus, other methods for treating type I allergic reactions are needed.

One approach to the treatment of type I allergic disorders has been theproduction of monoclonal antibodies which react with soluble (free) IgEin serum, block IgE from binding to its receptor on mast cells andbasophils, and do not bind to receptor-bound IgE (i.e., they arenon-anaphylactogenic). Two such monoclonal antibodies are in advancedstages of clinical development for treatment of IgE-mediated allergicreactions (see, e.g., Chang, T. W., 2000, Nat. Biotech. 18:157-162;incorporated herein by reference in its entirety).

One of the most promising treatments for IgE-mediated allergic reactionsis the active immunization against appropriate non-anaphylactogenicepitopes on endogenous IgE. Stanworth et al. (U.S. Pat. No. 5,601,821;incorporated herein by reference in its entirety) described a strategyinvolving the use of a peptide derived from the CεH4 domain of the humanIgE coupled to a heterologous carrier protein as an allergy vaccine.However, this peptide has been shown not to induce the production ofantibodies that react with native soluble IgE. Further, Hellman (U.S.Pat. No. 5,653,980) proposed anti-IgE vaccine compositions based onfusion of full length CεH2-CεH3 domains (approximately 220 amino acidlong) to a foreign carrier protein. However, the antibodies induced bythe anti-IgE vaccine compositions proposed in Hellman will most likelyit result in anaphylaxis since antibodies against some portions of theCεH2 and CεH3 domains of the IgE molecule have been shown to cross-linkthe IgE receptor on the surface of mast cell and basophils and lead toproduction of mediators of anaphylaxis (See, e.g., Stadler et al., 1993,Int. Arch. Allergy and Immunology 102:121-126; incorporated herein byreference in its entirety). Therefore, a need remains for treatment ofIgE-mediated allergic reactions which do not induce anaphylacticantibodies.

The significant concern over induction of anaphylaxis has resulted inthe development of another approach to the treatment of type I allergicdisorders consisting of mimotopes that could induce the production ofanti-IgE polyclonal antibodies when administered to animals (See, e.g.,Rudolf, et al., 1998, Journal of Immunology 160:3315-3321; incorporatedherein by reference in its entirety). Kricek et al. (InternationalPublication No. WO 97/31948; incorporated herein by reference in itsentirety) screened phage-displayed peptide libraries with the monoclonalantibody BSWI7 to identify peptide mimotopes that could mimic theconformation of the IgE receptor binding. These mimotopes couldpresumably be used to induce polyclonal antibodies that react with freenative IgE, but not with receptor-bound IgE as well as block IgE frombinding to its receptor. Kriek et al. disclosed peptide mimotopes thatare not homologous to any part of the IgE molecule and are thusdifferent from peptides disclosed in the present invention.

As evidenced by a survey of the art, there remains a need for enhancingthe therapeutic efficacy of current methods of treating or preventingdisorders such as cancer, autoimmune disease, inflammatory disorder, orallergy. In particular, there is a need for enhancing the effectorfunction, particularly, the cytotoxic effect of therapeutic antibodiesused in treatment of cancer. The current state of the art is alsolacking in treating or preventing allergy disorders (e.g., either byantibody therapy or vaccine therapy).

4. SUMMARY OF THE INVENTION

The instant invention provides humanized FcγRIIB antibodies, an isolatedantibody or a fragment thereof that specifically binds FcγRIIB,particularly human FcγRIIB, more particularly native human FcγRIIB, witha greater affinity than said antibody or a fragment thereof bindsFcγRIIA, particularly human FcγRIIA, more particularly native humanFcγRIIA. As used herein, “native FcγRIIB or FcγRIIA” means FcγRIIB orFcγRIIA which is endogenously expressed in a cell and is present on thecell surface of that cell or recombinantly expressed in a mammalian celland present on the cell surface, but is not FcγRIIB or FcγRIIA expressedin a bacterial cell or denatured, isolated FcγRIIB or FcγRIIA. Theinstant invention encompasses humanized antibodies, and antigen bindingfragments thereof, derived from antibodies that bind FcγRIIB,particularly human FcγRIIB, more particularly native human FcγRIIB, witha greater affinity than said antibody or a fragment thereof bindsFcγRIIA, particularly human FcγRIIA, more particularly native humanFcγRIIA. In most preferred embodiments, the instant invention relates tohumanized 2B6 or 3H7 antibodies or fragments thereof, preferably antigenbinding fragments thereof. In another preferred embodiments, theinvention relates to humanized 1D5, 2E1, 2H9, 2D11, or 1F2 antibodiesand fragments thereof, preferably antigen binding fragments thereof.

Preferably the humanized antibodies of the invention bind theextracellular domain of native human FcγRIIB. The humanized anti-FcγRIIBantibodies of the invention may have a heavy chain variable regioncomprising the amino acid sequence of a CDR1 (e.g., SEQ ID NO:1, SEQ IDNO:29, an amino acid sequence corresponding to amino acids 31-35 as setforth in SEQ ID NO:60, or an amino acid sequence corresponding to aminoacids 31-35 as set forth in SEQ ID NO:68) and/or a CDR2 (e.g., SEQ IDNO:2, SEQ ID NO:30, an amino acid sequence corresponding to amino acids50-66 as set forth in SEQ ID NO:60, or an amino acid sequencecorresponding to amino acids 50-66 as set forth in SEQ ID NO:68) and/ora CDR3 (e.g., SEQ ID NO:3, SEQ ID NO:31, an amino acid sequencecorresponding to amino acids 99-110 as set forth in SEQ ID NO:60, or anamino acid sequence corresponding to amino acids 99-110 as set forth inSEQ ID NO:68) and/or a light chain variable region comprising the aminoacid sequence of a CDR1 (e.g., SEQ ID NO:8, SEQ ID NO:38, or an aminoacid sequence corresponding to amino acids 24-34 as set forth in SEQ IDNO:62) and/or a CDR2 (e.g., SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:39, or an amino acid sequence corresponding to amino acids 50-56as set forth in SEQ ID NO:62) and/or a CDR3 (e.g., SEQ ID NO:12, SEQ IDNO:40, or an amino acid sequence corresponding to amino acids 89-97 asset forth in SEQ ID NO:62).

In other embodiments, the humanized antibodies of the invention comprisea light chain variable region comprising the amino acid sequence of SEQID NO. 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:46, or SEQ ID NO:62,and/or a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO:24, SEQ ID NO:37, SEQ ID NO:60, or SEQ ID NO:68 and/oramino acid sequence variants thereof. In a preferred embodiment, thehumanized antibodies of the invention comprise a light chain variableregion comprising the amino the amino acid sequence of SEQ ID NO:62 anda heavy chain variable region comprising the amino acid sequence of SEQID NO:68.

In particular, the invention provides a humanized antibody thatimmunospecifically binds to extracellular domain of native humanFcγRIIB, said antibody comprising (or alternatively, consisting of) a VHCDR1 and a VL CDR1; a VH CDR1 and a VL CDR2; a VH CDR1 and a VL CDR3; aVH CDR2 and a VL CDR1; VH CDR2 and VL CDR2; a VH CDR2 and a VL CDR3; aVH CDR3 and a VH CDR1; a VH CDR3 and a VL CDR2; a VH CDR3 and a VL CDR3;a VH CDR1, a VH CDR2 and a VL CDR1; a VH CDR1, a VH CDR2 and a VL CDR2;a VH CDR1, a VH CDR2 and a VL CDR3; a VH CDR2, a VH CDR3 and a VL CDR1,a VH CDR2, a VH CDR3 and a VL CDR2; a VH CDR2, a VH CDR2 and a VL CDR3;a VH CDR1, a VL CDR1 and a VL CDR2; a VH CDR1, a VL CDR1 and a VL CDR3;a VH CDR2, a VL CDR1 and a VL CDR2; a VH CDR2, a VL CDR1 and a VL CDR3;a VH CDR3, a VL CDR1 and a VL CDR2; a VH CDR3, a VL CDR1 and a VL CDR3;a VH CDR1, a VH CDR2, a VH CDR3 and a VL CDR1; a VH CDR1, a VH CDR2, aVH CDR3 and a VL CDR2; a VH CDR1, a VH CDR2, a VH CDR3 and a VL CDR3; aVH CDR1, a VH CDR2, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR2, a VLCDR1 and a VL CDR3; a VH CDR1, a VH CDR3, a VL CDR1 and a VL CDR2; a VHCDR1, a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR2, a VH CDR3, a VLCDR1 and a VL CDR2; a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR3; a VHCDR2, a VH CDR3, a VL CDR2 and a VL CDR3; a VH CDR1, a VH CDR2, a VHCDR3, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR2, a VH CDR3, a VLCDR1 and a VL CDR3; a VH CDR1, a VH CDR2, a VL CDR1, a VL CDR2, and a VLCDR3; a VH CDR1, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3; a VHCDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3; or any combinationthereof of the VH CDRs and VL CDRs disclosed herein.

In one specific embodiment, the invention provides a humanized 2B6antibody, wherein a VH region consists of the FR segments from the humangermline VH segment VH1-18 and JH6, and the CDR regions of the 2B6 VH,having the amino acid sequence of SEQ ID NO. 24. In another embodiment,the invention provides a humanized 2B6 antibody wherein a VH regioncomprises the amino acid sequence SEQ ID NO:60. In yet otherembodiments, the invention provides a humanized 2B6 antibody wherein aVH region comprises the amino acid sequence SEQ ID NO:68. In anotherspecific embodiment, the invention provided a humanized 2B6 antibodywherein a VL region consists of the FR segments of the human germline VLsegment VK-A26 and JK4 and the CDR regions of 2B6 VL, having an aminoacid sequence of SEQ ID NO. 18, SEQ ID NO. 20, or SEQ ID NO. 22. Inother embodiments, the invention provides a humanized 2B6 antibodywherein the VL region comprises the amino acid sequence SEQ ID NO:62.

In one specific embodiment, the invention provides a humanized 3H7antibody, wherein a VH region consists of the FR segments from a humangermline VH segment, and the CDR regions of 3H7 VH, having the aminoacid sequence of SEQ ID NO:37. In another specific embodiment, thehumanized 3H7 antibody further comprises a VL region, which consists ofthe FR segments of the human germline VL segment, and the CDR regions of3H7 VL, having an amino acid sequence of SEQ ID NO:46.

The present invention provides humanized antibody molecules specific forFcγRIIB in which one or more regions of one or more CDRs of the heavyand/or light chain variable regions of a human antibody (the recipientantibody) have been substituted by analogous parts of one or more CDRsof a donor monoclonal antibody which specifically binds FcγRIIB with agreater affinity than FcγRIIA, e.g., of the monoclonal antibody producedby clone 2B6 or 3H7 that bind FcγRIIB, having ATCC accession numbersPTA-4591 and PTA-4592, respectively, or a monoclonal antibody producedby clone 1D5, 2E1, 2H9, 2D11 or 1F2, having ATCC Accession numbers,PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively. In amost preferred embodiment, the humanized antibody can specifically bindto the same epitope as the donor murine antibody. It will be appreciatedby one skilled in the art that the invention encompasses CDR grafting ofantibodies in general. Thus, the donor and acceptor antibodies may bederived from animals of the same species and even same antibody class orsub-class. More usually, however, the donor and acceptor antibodies arederived from animals of different species. Typically the donor antibodyis a non-human antibody, such as a rodent MAb, and the acceptor antibodyis a human antibody.

In some embodiments, at least one CDR from the donor antibody is graftedonto the human antibody. In other embodiments, at least two andpreferably all three CDRs of each of the heavy and/or light chainvariable regions are grafted onto the human antibody. The CDRs maycomprise the Kabat CDRs, the structural loop CDRs (“Chothia CDRs”) or acombination thereof. In some embodiments, the invention encompasses ahumanized FcγRIIB antibody comprising at least one CDR grafted heavychain and at least one CDR-grafted light chain.

In a preferred embodiment, the CDR regions of the humanized FcγRIIBspecific antibody are derived from a murine antibody against FcγRIIB. Insome embodiments, the humanized antibodies described herein comprisealterations, including, but not limited to, amino acid deletions,insertions, and modifications, of the acceptor antibody, i.e., human,heavy and/or light chain variable domain framework regions that arenecessary for retaining an/or altering and/or improving bindingspecificity of the donor monoclonal antibody. Such modifications maymodify the amino acid sequence of the framework region to correspond tothe framework region of the donor, e.g. murine, antibody. In certainembodiments, the invention encompasses a humanized antibody havingphenylalanine at amino acid number 21 of the light chain variable domainframework region 1 (e.g., corresponding to amino acid number 21 of SEQID NO:62). In other embodiments, the invention encompasses a humanizedantibody having one or more of an isoleucine at amino acid number 13 ofthe heavy chain variable domain framework region 2 (e.g., correspondingto amino acid number 48 of SEQ ID NO:60), a valine at amino acid number6 of the heavy chain variable domain framework region 3 (e.g.,corresponding to amino acid number 72 of SEQ ID NO:60), a valine atamino acid number 7 of the heavy chain variable domain framework region3 (e.g., corresponding to amino acid number 73 of SEQ ID NO:60), avaline at amino acid number 8 of the heavy chain variable domainframework region 3 (e.g., corresponding to amino acid number 74 of SEQID NO:60), or any combination thereof. In still other embodiments, theinvention encompasses a humanized antibody having one or more of anisoleucine at amino acid number 13 of the heavy chain variable domainframework region 2 (e.g., corresponding to amino acid number 48 of SEQID NO:68), a valine at amino acid number 6 of the heavy chain variabledomain framework region 3 (e.g., corresponding to amino acid number 72of SEQ ID NO:68), an aspartic acid at amino acid number 7 of the heavychain variable domain framework region 3 (e.g., corresponding to aminoacid number 73 of SEQ ID NO:68), a threonine at amino acid number 8 ofthe heavy chain variable domain framework region 3 (e.g., correspondingto amino acid number 74 of SEQ ID NO:68), or any combination thereof. Incertain embodiments, the invention encompasses humanized antibodiescomprising at least one amino acid modification (e.g., insertion,deletion, substitution) in one or more of the light chain variabledomain framework regions. In certain embodiments, the inventionencompasses a humanized antibody comprising a modification at amino acid21 of the light chain variable domain framework region 1 (e.g.,corresponding to amino acid number 21 in SEQ ID NO:62), whichmodification is preferably a substitution with phenylalanine. In otherembodiments, the invention encompasses humanized antibodies comprisingat least one amino acid modification (e.g., insertion, deletion,substitution) in one or more of the heavy chain variable domainframework regions. In certain embodiments, the invention encompasses ahumanized antibody comprising a modification at amino acid 13 of theheavy chain variable domain framework region 2, which modification ispreferably a substitution with isoleucine (e.g., corresponding to aminoacid number 48 in SEQ ID NO:68), and/or a modification at amino acid 6of the heavy chain variable domain framework region 3, whichmodification is preferably a substitution with valine (e.g.,corresponding to amino acid number 72 in SEQ ID NO:68), and/or amodification at amino acid 7 of the heavy chain variable domainframework region 3, which modification is preferably a substitution withaspartic acid (e.g., corresponding to amino acid number 73 in SEQ IDNO:68), and/or a modification at amino acid 8 of the heavy chainvariable domain framework region 3, which modification is preferably asubstitution with threonine (e.g., corresponding to amino acid number 74in SEQ ID NO:68). In another specific example in accordance with thisembodiment, the invention encompasses a humanized antibody comprising amodification at amino acid 13 of the heavy chain variable domainframework region 2, which modification is preferably a substitution withisoleucine (e.g., corresponding to amino acid number 48 in SEQ IDNO:60), and/or a modification at amino acid 6 of the heavy chainvariable domain framework region 3, which modification is preferably asubstitution with valine (e.g., corresponding to amino acid number 72 inSEQ ID NO:60), and/or a modification at amino acid 7 of the heavy chainvariable domain framework region 3, which modification is preferably asubstitution with valine (e.g., corresponding to amino acid number 73 inSEQ ID NO:60), and/or a modification at amino acid 8 of the heavy chainvariable domain framework region 3, which modification is preferably asubstitution with valine (e.g., corresponding to amino acid number 74 inSEQ ID NO:60). Non-limiting examples of humanized 2B6 heavy chain andlight chain variable domains amino acid sequences are set forth in FIG.2A and FIG. 2B, respectively. The invention further encompasses anycombination of the foregoing amino acid modifications the heavy and orlight chain variable domain framework regions. In some embodiments, theframework regions of the humanized antibodies described herein do notnecessarily consist of the precise amino acid sequence of the frameworkregion of a natural occurring human antibody variable region, butcontain various alterations, including, but not limited to, amino aciddeletions, insertions, modifications that alter the property of thehumanized antibody, for example, improve the binding properties of ahumanized antibody region that is specific for the same target as themurine FcγRIIB specific antibody. In most preferred embodiments, aminimal number of alterations are made to the framework region in orderto avoid large-scale introductions of non-human framework residues andto ensure minimal immunogenicity of the humanized antibody in humans. Insome embodiments, the framework residues are derived from the humangermline VH segment VH1-18 and JH6 and/or the human germline VL segmentVK-A26 and JK4. In certain embodiments of the invention, there are noalterations made to the framework regions. In specific embodiments, thedonor monoclonal antibody of the present invention is a monoclonalantibody produced by clone 2B6 or 3H7 which binds FcγRIIB, having ATCCaccession number PTA-4591 and PTA-4592, respectively, or a monoclonalantibody produced by clone 1D5, 2E1, 2H9, 2D11 or 1F2 having ATCCAccession number PTA-5958, PTA-5961, PTA-5962, PTA-5960 and PTA-5959,respectively.

The humanized antibodies of the present invention include completeantibody molecules having full length heavy and light chains, or anyfragment thereof, such as the Fab or (Fab′)₂ fragments, a heavy chainand light chain dimer, or any minimal fragment thereof such as an Fv, anSCA (single chain antibody), and the like that exhibits immunospecificbinding for the FcγRIIB.

In certain embodiments, the Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that the modifiedFc region has an altered binding affinity to a Fc receptor. In aspecific embodiment, the amino acid modification of the Fc regions ofthe humanized antibodies of the invention relative to a wild-type Fcregion comprise a substitution at position 243, 292, 300, 305 and 396.In a certain embodiment, the amino acid modification of the Fc regionsof the humanized antibodies of the invention relative to a wild-type Fcregion comprise a substitution at position 243 with leucine, at position292 with proline, at position 300 with leucine, at position 305 withisoleucine and at position 396 with leucine. In other embodiments, theFc domain of the antibody or fragment thereof has an increased bindingaffinity to FcγRIIB and/or FcγRIII relative to that of the wild-typeantibody.

In a specific embodiment, the Fc region of the humanized antibody of theinvention comprises a leucine at position 243, a proline at position292, a leucine at position 300, an isoleucine at position 305 and aleucine at position 396.

In a specific embodiment, a humanized 2B6 antibody of the inventioncomprises a heavy chain having the amino acid sequence SEQ ID NO:64. Inanother specific embodiment, a humanized 2B6 antibody of the inventioncomprises a heavy chain having the amino acid sequence SEQ ID NO:70. Instill other embodiments, the humanized 2B6 antibody comprises a lightchain having the amino acid sequence SEQ ID NO:66. In a preferredembodiment, a humanized 2B6 antibody of the invention comprises a heavychain containing the amino acid sequence SEQ ID NO:70 and a light chaincontaining the sequence SEQ ID NO:66. In a specific aspect of theinvention, plasmid pMGx0675 comprises the nucleotide sequences SEQ IDNO:69 and SEQ ID NO:65 that encode the heavy chain amino acid sequenceSEQ ID NO:70 and the light chain amino acid sequence SEQ ID NO:66,respectively. Plasmid pMGx0675 been deposited with the American TypeCulture Collection (10801 University Blvd., Manassas, Va. 20110-2209) onMay 23, 2006 under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedures, and assigned accession number PTA-7609,and is incorporated herein by reference.

In a particular embodiment, the invention relates to an isolatedantibody or a fragment thereof that specifically binds FcγRIIB via thevariable domain with a greater affinity than said antibody or a fragmentthereof binds FcγRIIA, and the constant domain of said antibody furtherhas an enhanced affinity for at least one or more Fc activationreceptors relative to an isotype control antibody. In yet anotherspecific embodiment, said Fc activation receptor is FcγRIIIA or FcγRIIA.

The invention encompasses methods for the production of antibodies ofthe invention or fragments thereof, particularly for the production ofhumanized anti-FcγRIIB specific antibodies, such that the FcγRIIBspecific antibodies have an enhanced specificity for FcγRIIB relative toFcγRIIA. The invention encompasses any method known in the art usefulfor the production of polypeptides, e.g., in vitro synthesis,recombinant DNA production, and the like. Preferably, the humanizedantibodies are produced by recombinant DNA technology. The humanizedFcγRIIB specific antibodies of the invention may be produced usingrecombinant immunoglobulin expression technology. Exemplary methods forthe production of recombinant humanized antibodies of the invention maycomprise the following: a) constructing, by conventional molecularbiology methods, an expression vector comprising an operon that encodesan antibody heavy chain in which the CDRs and a minimal portion of thevariable region framework that are required to retain donor antibodybinding specificity are derived from a non-human immunoglobulin, such asthe murine FcγRIIB specific monoclonal antibody, e.g., the monoclonalantibody produced by clone 2B6 or 3H7 which binds FcγRIIB, having ATCCaccession numbers PTA-4591 and PTA-4592, respectively, or the monoclonalantibody produced by clone 1D5, 2E1, 2H9, 2D11 or 1F2 having ATCCAccession number PTA-5958, PTA-5961, PTA-5962, PTA-5960 and PTA-5959,respectively, and the remainder of the antibody is derived from a humanimmunoglobulin, thereby producing a vector for the expression of ahumanized antibody heavy chain, e.g., the plasmid pMGx0675 comprisingnucleic acid sequence SEQ ID NO:69 encoding the amino acid sequence SEQID NO:70, said plasmid having the ATCC Accession number PTA-7609,deposited May 23, 2006; b) constructing, by conventional molecularbiology methods, an expression vector comprising an operon that encodesan antibody light chain in which the CDRs and a minimal portion of thevariable region framework that are required to retain donor antibodybinding specificity are derived from a non-human immunoglobulin, such asthe murine FcγRIIB monoclonal antibody, e.g., the monoclonal antibodyproduced by clone 2B6 or 3H7 which binds FcγRIIB, having ATCC accessionnumbers PTA-4591 and PTA-4592, respectively, or the monoclonal antibodyproduced by clone 1D5, 2E1, 2H9, 2D11 or 1F2 having ATCC Accessionnumber PTA-5958, PTA-5961, PTA-5962, PTA-5960 and PTA-5959,respectively, and the remainder of the antibody is derived from a humanimmunoglobulin, thereby producing a vector for the expression ofhumanized antibody light chain, e.g., the plasmid pMGx0675 comprisingnucleic acid sequence SEQ ID NO:65 encoding the amino acid sequence SEQID NO:66, said plasmid having the ATCC Accession number PTA-7609,deposited May 23, 2006; c) transferring the expression vectors to a hostcell by conventional molecular biology methods to produce a transfectedhost cell for the expression of humanized anti-FcγRIIB antibodies; d)culturing the transfected cell by conventional cell culture techniquesso as to produce humanized anti-FcγRIIB antibodies; and e) recovery ofthe anti-FcγRIIB antibodies from the culture by conventional means. Hostcells may be cotransfected with two expression vectors of the invention,the first vector containing an operon encoding a heavy chain derivedpolypeptide and the second containing an operon encoding a light chainderived polypeptide. The two vectors may contain different selectablemarkers but, with the exception of the heavy and light chain codingsequences, are preferably identical. This procedure provides for equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides, e.g., the plasmid pMGx0675, having ATCC Accession numberPTA-7609, deposited May 23, 2006, which comprises the nucleic acidsequences SEQ ID NO:69 and SEQ ID NO:65 that encode the amino acidsequences of the heavy chain and light chain of a humanized 2B6 antibody(i.e., SEQ ID NO:70 and SEQ ID NO:66, respectively). The codingsequences for the heavy and light chains may comprise cDNA or genomicDNA or both. The host cell used to express the recombinant antibody ofthe invention may be either a bacterial cell such as Escherichia coli,or, preferably, a eukaryotic cell. Preferably, a mammalian cell such asa Chinese hamster ovary cell or HEK-293 may be used. The choice ofexpression vector is dependent upon the choice of host cell, and may beselected so as to have the desired expression and regulatorycharacteristics in the selected host cell. The general methods forconstruction of the vector of the invention, transfection of cells toproduce the host cell of the invention, culture of cells to produce theantibody of the invention are all conventional molecular biologymethods. Likewise, once produced, the recombinant antibodies of theinvention may be purified by standard procedures of the art, includingcross-flow filtration, ammonium sulphate precipitation, affinity columnchromatography, gel electrophoresis and the like.

In some embodiments, cell fusion methods for making monoclonalantibodies may be used in the methods of the invention such as thosedisclosed in U.S. Pat. No. 5,916,771, incorporated herein by referencein its entirety. Briefly, according to this method, DNA encoding thedesired heavy chain (or a fragment of the heavy chain) is introducedinto a first mammalian host cell, while DNA encoding the desired lightchain (or a fragment of the light chain) is introduced into a secondmammalian host cell. The first transformed host cell and the secondtransformed host cell are then combined by cell fusion to form a thirdcell. Prior to fusion of the first and second cells, the transformedcells may be selected for specifically desired characteristics, e.g.,high levels of expression. After fusion, the resulting hybrid cellcontains and expresses both the DNA encoding the desired heavy chain andthe DNA encoding the desired light chain, resulting in production of themultimeric antibody.

The invention encompasses using the humanized antibodies of the presentinvention in conjunction with, or attached to, other antibodies orfragments thereof such as human or humanized monoclonal antibodies.These other antibodies may be reactive with other markers (epitopes)characteristic for the disease against which the antibodies of theinvention are directed or may have different specificities chosen, forexample, to recruit molecules or cells of the human immune system to thediseased cells. The antibodies of the invention (or parts thereof) maybe administered with such antibodies (or parts thereof) as separatelyadministered compositions or as a single composition with the two agentslinked by conventional chemical or by molecular biological methods.Additionally the diagnostic and therapeutic value of the antibodies ofthe invention may be augmented by labeling the humanized antibodies withlabels that produce a detectable signal (either in vitro or in vivo) orwith a label having a therapeutic property. Some labels, e.g.,radionuclides may produce a detectable signal and have a therapeuticproperty. Examples of radionuclide labels include ¹²⁵I, ¹³¹I, ¹⁴C.Examples of other detectable labels include a fluorescent chromophoresuch as fluorescein, phycobiliprotein or tetraethyl rhodamine forfluorescence microscopy; an enzyme which produces a fluorescent orcolored product for detection by fluorescence, absorbance, visible coloror agglutination, or which produces an electron dense product fordemonstration by electron microscopy; or an electron dense molecule suchas ferritin, peroxidase or gold beads for direct or indirect electronmicroscopic visualization. Labels having therapeutic properties includedrugs for the treatment of cancer, such as methotrexate and the like.

The methods of the invention also encompass polynucleotides that encodethe humanized antibodies of the invention or fragments thereof. In oneembodiment, the invention provides an isolated nucleic acid comprising anucleotide sequence encoding the heavy chain variable domain and/or thelight chain variable domain of an antibody of the invention. In aspecific embodiment, the invention provides an isolated nucleic acidcomprising a nucleotide sequence encoding a heavy chain variable domainhaving the amino acid sequence SEQ ID NO:60. In accordance with thisembodiment, the nucleic acid comprising the nucleotide sequence encodingthe heavy chain variable domain, amino acid sequence SEQ ID NO:60, may,for example, comprise the nucleotide sequence SEQ ID NO:59. In anotherspecific embodiment, the invention provides an isolated nucleic acidcomprising a nucleotide sequence encoding a heavy chain variable domainhaving the amino acid sequence SEQ ID NO:68. In accordance with thisembodiment, the nucleic acid comprising the nucleotide sequence encodingthe heavy chain variable domain, amino acid sequence SEQ ID NO:68, may,for example, comprise the nucleotide sequence SEQ ID NO:67. In stillother embodiments, the invention provides an isolated nucleic acidcomprising a nucleotide sequence encoding a light chain variable domainhaving the amino acid sequence SEQ ID NO:62. In accordance with theseembodiments, the nucleic acid comprising the nucleotide sequenceencoding the light chain variable domain, amino acid sequence SEQ IDNO:62, may, for example, comprise the nucleotide sequence SEQ ID NO:61.In yet other embodiments, the invention provides an isolated nucleicacid comprising a nucleotide sequence encoding a heavy chain or a lightchain of an antibody or a fragment thereof that specifically bindsFcγRIIB with greater affinity than said antibody or a fragment thereofbinds FcγRIIA. In another embodiment, the invention provides an isolatednucleic acid comprising a nucleotide sequence encoding a heavy chain ora light chain of an antibody or a fragment thereof that specificallybinds FcγRIIB and blocks the Fc binding domain of FcγRIIB. The inventionalso relates to a vector comprising said nucleic acid. The inventionfurther provides a vector comprising a first nucleic acid moleculecomprising a nucleotide sequence encoding a heavy chain and a secondnucleic acid molecule comprising a nucleotide sequence encoding a lightchain, said heavy chain and light chain being of an antibody or afragment thereof that specifically binds FcγRIIB with greater affinitythan said antibody or a fragment thereof binds FcγRIIA. In one specificembodiment, said vector is an expression vector. The invention furtherprovides host cells containing the vectors or nucleotide sequencesencoding the antibodies of the invention. In specific embodiments, theinvention encompasses nucleotide sequences encoding heavy and lightchains of the antibodies produced by the deposited hybridoma cloneshaving ATCC accession numbers PTA-4591, PTA-4592, PTA-5958, PTA-5961,PTA-5962, PTA-5960, and PTA-5959, or portions and/orvariants/derivatives thereof (e.g., CDRs, variable domains, etc. andhumanized versions thereof). In a specific embodiment, the inventionprovides the nucleotide sequence SEQ ID NO:63, which encodes a h2B6heavy chain, amino acid sequence SEQ ID NO:64. In another specificembodiment, the invention provides the nucleotide sequence SEQ ID NO:69,which encodes a h2B6 heavy chain, amino acid sequence SEQ ID NO:70. Instill another specific embodiment, the invention provides the nucleotidesequence SEQ ID NO:65, which encodes a h2B6 light chain, amino acidsequence SEQ ID NO:66. In preferred embodiments, the inventionencompasses nucleotide sequences encoding a heavy chain and/or lightchain having the amino acid sequences of SEQ ID NO:70 and SEQ ID NO:66,respectively.

The invention encompasses the use of the humanized antibodies of theinvention to detect the presence of FcγRIIB specifically (i.e., FcγRIIBand not FcγRIIA) in a biological sample.

Activating and inhibitory Fc receptors, e.g., FcγRIIA and FcγRIIB, arecritical for the balanced function of these receptors and propercellular immune responses. The invention encompasses the use of thehumanized antibodies of the invention for the treatment of any diseaserelated to loss of such balance and regulated control in the Fc receptorsignaling pathway. Thus, the humanized FcγRIIB antibodies of theinvention have uses in regulating the immune response, e.g., ininhibiting immune response in connection with autoimmune or inflammatorydisease, or allergic response. The humanized FcγRIIB antibodies of theinvention can also be used to alter certain effector functions toenhance, for example, therapeutic antibody-mediated cytotoxicity.

The humanized antibodies of the invention are useful for prevention ortreatment of cancer, for example, in one embodiment, as a single agenttherapy. In one embodiment of the invention, the humanized antibodies ofthe invention are useful for prevention or treatment of B-cellmalignancies, particularly non-Hodgkin's lymphoma or chronic lymphocyticleukemia. In particular embodiments, the cancer of the subject isrefractory to one or more standard or experimental therapies,particularly, to Rituxan treatment. The methods of the invention may beused for the treatment, management, prevention, or amelioration ofB-cell diseases, such as, B-cell chronic lymphocytic leukemia (B-CLL),non-Hodgkin's lymphoma, diffuse large B cell lymphoma, follicularlymphoma with areas of diffuse large B cell lymphoma, small lymphocyticlymphoma, mantle cell lymphoma, and diffuse small cleaved cell lymphoma.

In one embodiment, the invention provides for the use of a humanizedFcγRIIB-specific antibody in combination with a standard or experimentaltreatment regimen for B-cell malignancies (e.g., chemotherapy,radioimmunotherapy, or radiotherapy). Such combination therapy mayenhance the efficacy of standard or experimental treatment. Examples oftherapeutic agents that are particularly useful in combination with aFcγRIIB-specific antibody or an antigen-binding fragment thereof, forthe prevention, treatment, management, or amelioration of B-cellmalignancies, include, but are not limited to, Rituxan,interferon-alpha, and anti-cancer agents. Chemotherapeutic agents thatcan be used in combination with a FcγRIIB-specific antibody or anantigen-binding fragment thereof, include, but are not limited toalkylating agents, antimetabolites, natural products, and hormones. Thecombination therapies of the invention enable lower dosages of ananti-FcγRIIB antibody or an antigen-binding fragment thereof and/or lessfrequent administration of anti-FcγRIIB antibody or an antigen-bindingfragment thereof to a subject with a B-cell malignancy, to achieve atherapeutic or prophylactic effect.

In another embodiment, the use of a humanized FcγRIIB antibody or anantigen-binding fragment thereof prolongs the survival of a subjectdiagnosed with a B-cell malignancy.

In a preferred embodiment, the humanized antibodies of the invention areused for the treatment and/or prevention of melanoma. In anotherembodiment, the humanized antibodies are useful for prevention and/ortreatment of cancer, particularly in potentiating the cytotoxic activityof cancer antigen-specific therapeutic antibodies with cytotoxicactivity to enhance tumor cell killing and/or enhancing antibodydependent cytotoxic cellular (“ADCC”) activity, complement dependentcytotoxic (“CDC”) activity, or phagocytosis of the therapeuticantibodies.

The invention provides a method of treating cancer in a patient having acancer characterized by a cancer antigen, said method comprisingadministering to said patient a therapeutically effective amount of afirst humanized antibody or a fragment thereof that specifically bindsFcγRIIB with greater affinity than said antibody or a fragment thereofbinds FcγRIIA, and a second antibody that specifically binds said cancerantigen and is cytotoxic. The invention also provides a method oftreating cancer in a patient having a cancer characterized by a cancerantigen, said method comprising administering to said patient atherapeutically effective amount of an humanized antibody or a fragmentthereof that specifically binds FcγRIIB, particularly native humanFcγRIIB, with greater affinity than said antibody or a fragment thereofbinds FcγRIIA, preferably native human FcγRIIA, and the constant domainof which further has an increased affinity for one or more Fc activationreceptors, when the antibody is monomeric, such as FcγRIIIA, and anantibody that specifically binds said cancer antigen and is cytotoxic.In one particular embodiment, said Fc activation receptor is FcγRIIIA

In some embodiments, the invention encompasses antibodies comprisingvariant Fc regions that bind FcRn with an enhanced affinity, resultingin an increased antibody half life, e.g., a half-life of greater than 15days, preferably greater than 20 days, greater than 25 days, greaterthan 30 days, greater than 35 days, greater than 40 days, greater than45 days, greater than 2 months, greater than 3 months, greater than 4months, or greater than 5 months. Although not intending to be bound bya particular mechanism of action the neonatal Fc receptor (FcRn) playsan important role in regulating the serum half-lives of IgG antibodies.A correlation has been established between the pH-dependent bindingaffinity of IgG antibodies to FcRn and their serum half-lives in mice.The increased half-lives of the antibodies of the present invention orfragments thereof in a mammal, preferably a human, results in a higherserum titer of said antibodies or antibody fragments in the mammal, andthus, reduces the frequency of the administration of said antibodies orantibody fragments and/or reduces the concentration of said antibodiesor antibody fragments to be administered. For example, antibodies orfragments thereof with increased in vivo half-lives can be generated bymodifying (e.g., substituting, deleting or adding) amino acid residuesidentified as involved in the interaction between the Fc domain and theFcRn receptor. For example, the invention encompasses antibodiescomprising variant Fc regions which have at least one or moremodification that enhances the affinity to FcRn, e.g., a modification ofone or more amino acid residues 251-256, 285-290, 308-314, 385-389, and428-436, or a modification at positions 250 and 428, see, e.g., Hintonet al., 2004, J. Biol. Chem. 279(8): 6213-6; PCT Publication No. WO97/34631; and WO 02/060919, all of which are incorporated herein byreference in their entirety.

In yet another embodiment, the invention provides a method of regulatingimmune-complex mediated cell activation in a patient, said methodcomprising administering to said patient a therapeutically effectiveamount of an antibody or fragment thereof that specifically binds theextracellular domain of human FcγRIIB and blocks the Fc binding site ofhuman FcγRIIB. In a preferred embodiment, administration of the antibodyor fragment thereof results in an enhanced immune response, such as anincrease in an antibody-dependent cellular response. In anotherpreferred embodiment, the immune complex mediated cell activation is Bcell activation, mast cell activation, dendritic cell activation ormacrophage activation.

In another embodiment, the invention provides a method of breakingtolerance to an antigen in a patient, said method comprisingadministering to a patient in need thereof (1) an antigen-antibodycomplex comprising said antigen and (2) an antibody or fragment thereofthat specifically binds the extracellular domain of human FcγRIIB andblocks the Fc binding site of human FcγRIIB, thereby breaking tolerancein said patient to said antigen. The antibody or fragment thereof can beadministered before, concurrently with, or after administration of saidantigen-antibody complex.

In another embodiment, the invention provides a method of enhancing anantibody mediated cytotoxic effect in a subject being treated with acytotoxic antibody, said method comprising administering to said patienta humanized antibody of the invention, or a fragment thereof, in anamount sufficient to enhance the cytotoxic effect of said cytotoxicantibody. In yet another embodiment, the invention provides a method ofenhancing an antibody-mediated cytotoxic effect in a subject beingtreated with a cytotoxic antibody, said method comprising administeringto said patient a humanized antibody of the invention, or a fragmentthereof, further having an enhanced affinity for an Fc inhibitoryreceptor, when monomeric, in an amount sufficient to enhance thecytotoxic effect of said cytotoxic antibody. In yet another embodiment,the invention provides a method further comprising the administration ofone or more additional cancer therapies.

The invention encompasses the use of the humanized antibodies of theinvention in combination with any therapeutic antibody that mediates itstherapeutic effect through cell killing to potentiate the antibody'stherapeutic activity. In one particular embodiment, the humanizedantibodies of the invention potentiate the antibody's therapeuticactivity by enhancing antibody-mediated effector function. In anotherembodiment of the invention, the humanized antibodies of the inventionpotentiate the cytotoxic antibody's therapeutic activity by enhancingphagocytosis and opsonization of the targeted tumor cells. In yetanother embodiment of the invention, the humanized antibodies of theinvention potentiate the antibody's therapeutic activity by enhancingantibody-dependent cell-mediated cytotoxicity (“ADCC”) in destruction ofthe targeted tumor cells. In certain embodiments, the antibodies of theinvention are used in combination with Fc fusion proteins to enhanceADCC.

Although not intending to be bound by a particular mechanism of action,the combination of a humanized antibody of the invention in combinationwith a therapeutic antibody has an enhanced therapeutic effect due, inpart, to the cytotoxic ability of the FcγRIIB specific humanizedantibody to eliminate macrophages expressing the inhibitory Cribreceptors. Therefore, there is a higher concentration of cellsexpressing activating Fig receptors remaining per dose of thetherapeutic antibody.

In some embodiments, the invention encompasses use of the humanizedantibodies of the invention in combination with a therapeutic antibodythat does not mediate its therapeutic effect through cell killing topotentiate the antibody's therapeutic activity. In a specificembodiment, the invention encompasses use of the humanized antibodies ofthe invention in combination with a therapeutic apoptosis inducingantibody with agonistic activity, e.g., anti-Fas antibody. Therapeuticapoptosis inducing antibodies may be specific for any death receptorknown in the art for the modulation of apoptotic pathway, e.g., TNFRreceptor family member or a TRAIL family member.

The invention encompasses using the humanized antibodies of theinvention to block macrophage mediated tumor cell progression andmetastasis. The humanized antibodies of the invention are particularlyuseful in the treatment of solid tumors, where macrophage infiltrationoccurs. The antagonistic humanized antibodies of the invention areparticularly useful for controlling, e.g., reducing or eliminating,tumor cell metastasis, by reducing or eliminating the population ofmacrophages that are localized at the tumor site. The invention furtherencompasses humanized antibodies that effectively deplete or eliminateimmune effector cells other than macrophages that express FcγRIIB, e.g.,dendritic cells. Effective depletion or elimination of immune effectorcells using the antibodies of the invention may range from a reductionin population of the effector cells by 50%, 60%, 70%, 80%, preferably90%, and most preferably 99%.

In some embodiments, the invention encompasses use of the humanizedantibodies of the invention in combination with therapeutic antibodiesthat immunospecifically bind to tumor antigens that are not expressed onthe tumor cells themselves, but rather on the surrounding reactive andtumor supporting, non-malignant cells comprising the tumor stroma. In apreferred embodiment, a humanized antibody of the invention is used incombination with an antibody that immunospecifically binds a tumorantigen on a fibroblast cell, e.g., fibroblast activation protein (FAP).

The invention provides a method of treating an autoimmune disorder in apatient in need thereof, said method comprising administering to saidpatient a therapeutically effective amount of one or more humanizedantibodies of the invention. The invention also provides a method oftreating an autoimmune disorder in a patient in need thereof, saidmethod further comprising administering to said patient atherapeutically effective amount of one or more anti-inflammatoryagents, and/or one or more immunomodulatory agents. The inventionprovides methods of treating or ameliorating the symptoms of autoimmunediseases including, but not limited to, Type I Diabetes, psoriasis,rheumatoid arthritis, lupus (particularly, cutaneous), inflammatorybowel disease (IBD), Crohn's disease, ulcerative colitis, multiplesclerosis. In certain embodiments, the he methods of the invention arefor use in subjects with early stage disease to slow or reduce thedamage from the autoimmunity and maintain a high level of functionand/or reduce the need for or prevent an increase in the level of othertherapy, e.g., administrations of an immunosuppressants or ananti-inflammatory.

The invention also provides a method of treating an inflammatorydisorder in a patient in need thereof, said method comprisingadministering to said patient a therapeutically effective amount of oneor more humanized antibodies of the invention. The invention alsoprovides a method of treating an inflammatory disorder in a patient inneed thereof, said method further comprising administering to saidpatient a therapeutically effective amount of one or moreanti-inflammatory agents, and/or one or more immunomodulatory agents.

The invention provides a method of enhancing an immune response to avaccine composition in a subject, said method comprising administeringto said subject a humanized antibody or an antigen-binding fragmentthereof that specifically binds FcγRIIB with greater affinity than saidantibody or a fragment thereof binds FcγRIIA, and a vaccine composition,such that said antibody or a fragment thereof is administered in anamount effective to enhance the immune response to said vaccinecomposition in said subject. The humanized antibodies of the inventionmay be used to enhance a humoral and/or cell mediated response againstthe antigen(s) of the vaccine composition. The antibodies of theinvention may be used in combination with any vaccines known in the art.The invention encompasses the use of the humanized antibodies of theinvention to either prevent or treat a particular disorder, where anenhanced immune response against a particular antigen or antigens iseffective to treat or prevent the disease or disorder.

The invention also provides a method for enhancing immune therapy for aninfectious agent wherein the humanized antibodies of the invention areadministered to a patient that is already infected by a pathogen, suchas HIV, HCV or HSV, to enhance opsonization and phagocytosis of infectedcells. In yet other embodiments, the invention encompasses method fortreating sepsis or septic shock using the humanized antibodies of theinvention. The role of FcγRIIB in sepsis has been described inClatworthy et al., 2004, J Exp Med 199:717-723; incorporated herein byreference in its entirety.

The invention provides a method of treating diseases with impairedapoptotic mediated signaling, e.g., cancer, autoimmune disease In aspecific embodiment, the invention encompasses a method of treating adisease with deficient Fas-mediated apoptosis, said method comprisingadministering a humanized antibody of the invention in combination withan anti-Fas antibody.

The invention further provides a method for treating or preventing anIgE-mediated allergic disorder in a patient in need thereof, comprisingadministering to said patient a therapeutically effective amount of thehumanized agonistic antibodies of the invention. The invention alsoprovides a method for treating or preventing an IgE-mediated allergicdisorder in a patient in need thereof, comprising administering to saidpatient the humanized antibodies of the invention in combination withother therapeutic antibodies or vaccine compositions used for thetreatment or prevention of IgE-mediated allergic disorders.

In another embodiment, the invention provides for the use of aFcγRIIB-specific antibody conjugated to a therapeutic agent or drug.Examples of therapeutic agents which may be conjugated to ananti-FcγRIIB antibody or an antigen-binding fragment thereof include,but are not limited to, cytokines, toxins, radioactive elements, andantimetabolites.

In another embodiment, the invention provides a method of diagnosis ofan autoimmune disease in a subject comprising: (i) contacting abiological sample from said subject with an effective amount of ahumanized antibody of the invention; and (ii) detecting binding of saidhumanized antibody or a fragment thereof, wherein detection of saiddetectable marker above a background or standard level indicates thatsaid subject has an autoimmune disease.

The invention further provides a pharmaceutical composition comprising(i) a therapeutically effective amount of a humanized antibody or afragment thereof that specifically binds FcγRIIB with greater affinitythan said antibody or a fragment thereof binds FcγRIIA; and (ii) apharmaceutically acceptable carrier. The invention additionally providesa pharmaceutical composition comprising (i) a therapeutically effectiveamount of a humanized antibody or a fragment thereof that specificallybinds FcγRIIB with greater affinity than said antibody or a fragmentthereof binds FcγRIIA; (ii) a cytotoxic antibody that specifically bindsa cancer antigen; and (iii) a pharmaceutically acceptable carrier. In aspecific embodiment, the antibody or fragment thereof specifically bindsthe extracellular domain of human FcγRIIB, blocks the Fc binding site ofhuman FcγRIIB, and also blocks crosslinking of FcγRIIB to a Fc receptor.In a specific embodiment, Fc region of the antibody of the inventioncomprises one or more of an alanine at position 265, a glutamine atposition 297, leucine at position 243, a proline at position 292, aleucine at position 300, an isoleucine at position 305, or a leucine atposition 396. the In yet another embodiment, the antibody or fragmentthereof that specifically binds the extracellular domain of humanFcγRIIB and blocks the Fc binding site of human FcγRIIB comprises a Fcregion comprising at least one amino acid modification relative to awild-type Fc region, such that the modified Fc region has an alteredbinding affinity to a Fc receptor. In a specific embodiment, the aminoacid modification comprises a substitution at position 265 or 297,preferably a substitution at position 265 with alanine or a substitutionat position 297 with glutamine. In another embodiment, the amino acidmodification comprises a substitution at position 243, 292, 300, 305 and396, preferably a substitution at 243 with leucine, a substitution atposition 292 with proline, a substitution at position 300 with leucine,a substitution at position 305 with isoleucine, and a substitution atposition 396 with leucine.

The invention also provides combination therapy methods. The methods ofthe invention can be carried out in combination with any standardtreatment for the particular indication, e.g., in the case of treatmentof cancer, standard chemotherpiy and/or anti-angiogenic agents and, inthe case of the treatment of an autoimmune disorder, standardimmunosuppressant and/or anti-inflammatory treatments. The antibodiesand/or compositions of the invention may be administered with othertherapies such as anti-inflammatory agents, steroidal therapies (forexample, but not limited to, glucocorticoids, dexamethasone, cortisone,hydrocortisone, prednisone, prednisolone, triamcinolone, azulfidine,etc.), non-steroidal anti-inflammatories (NSAIDS) (for example, but notlimited to, COX-2 inhibitors, aspirin, ibuprofen, diclofenac, etodolac,fenoprofen, indomethacin, ketolorac, oxaprozin, nabumetone, sulindac,tolmentin, naproxen, ketoprofen, etc.), beta-agonists, anti-cholinergicagents, immunomodulatory agents (for example, but not limited to, T cellreceptor modulators, cytokine receptor modulators, T cell depletingagents, cytokine antagonists, monokine agonists, lymphokine inhibitors,etc.), immunosuppressants (such as, but not limited to, methotrexate orcyclosporin), anti-angiogenic agents (e.g., angiostatin and TNF-.alpha.antagonists and/or inhibitors (for example, but not limited to,etanercept and infliximab)), dapsone and psoralens. In certainembodiments of the invention, subjects which have become refractory toconventional treatments are treated using methods of the invention.

In certain embodiments of the invention, pharmaceutical compositions areprovided for use in accordance with the methods of the invention, saidpharmaceutical compositions comprising a humanized FcγRIIB antibody oran antigen-binding fragment thereof, in an amount effective to prevent,treat, manage, or ameliorate a B-cell malignancy, or one or moresymptoms thereof, and a pharmaceutically acceptable carrier. Theinvention also provides pharmaceutical compositions for use inaccordance with the methods of the invention, said pharmaceuticalcompositions comprising a humanized FcγRIIB antibody or anantigen-binding fragment thereof, a prophylactic or therapeutic agentother than a FcγRIIB antagonist, and a pharmaceutically acceptablecarrier.

The antibodies and/or compositions of the invention may be administeredparenterally, for example, intravenously, intramuscularly orsubcutaneously, or, alternatively, may be administered orally. Theantibodies and/or compostions of the invention may also be administeredas a sustained release formulation.

4.1 Definitions

As used herein, the term “specifically binds to FcγRIIB” and analogousterms refer to antibodies or fragments thereof (or any other FcγRIIBbinding molecules) that specifically bind to FcγRIIB or a fragmentthereof and do not specifically bind to other Fc receptors, inparticular to FcγRIIA. Further it is understood to one skilled in theart, that an antibody that specifically binds to FcγRIIB, may bindthrough the variable domain or the constant domain of the antibody. Ifthe antibody that specifically binds to FcγRIIB binds through itsvariable domain, it is understood to one skilled in the art that it isnot aggregated, i.e., is monomeric. An antibody that specifically bindsto FcγRIIB may bind to other peptides or polypeptides with loweraffinity as determined by, e.g., immunoassays, BIAcore, or other assaysknown in the art. Preferably, antibodies or fragments that specificallybind to FcγRIIB or a fragment thereof do not cross-react with otherantigens. Antibodies or fragments that specifically bind to FcγRIIB canbe identified, for example, by immunoassays, BIAcore, or othertechniques known to those of skill in the art. An antibody or a fragmentthereof binds specifically to a FcγRIIB when it binds to FcγRIIB withhigher affinity than to any cross-reactive antigen as determined usingexperimental techniques, such as western blots, radioimmunoassays (RIA)and enzyme-linked immunosorbent assays (ELISAs). See, e.g., Paul, ed.,1989, Fundamental Immunology Second Edition, Raven Press, New York atpages 332-336 for a discussion regarding antibody specificity(incorporated herein by reference in its entirety).

As used herein, the term “native FcγRIIB” refers to FcγRIIB which isendogenously expressed and present on the surface of a cell. In someembodiments, “native FcγRIIB” encompasses a protein that isrecombinantly expressed in a mammalian cell. Preferably, the nativeFcγRIIB is not expressed in a bacterial cell, i.e., E. coli. Mostpreferably the native FcγRIIB is not denatured, i.e., it is in itsbiologically active conformation.

As used herein, the term “endogenous” in the context of a cellularprotein refers to protein naturally occurring and/or expressed by thecell in the absence of recombinant manipulation; accordingly, the terms“endogenously expressed protein” or “endogenous protein” excludescellular proteins expressed by means of recombinant technology.

As used herein, the terms “antibody” and “antibodies” refer tomonoclonal antibodies, multispecific antibodies, human antibodies,humanized antibodies, synthetic antibodies, chimeric antibodies,camelized antibodies, single-chain Fvs (scFv), single chain antibodies,Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFv),intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g.,anti-Id and anti-anti-Id antibodies to antibodies of the invention), andepitope-binding fragments of any of the above. In particular, antibodiesinclude immunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site Immunoglobulin molecules can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁and IgA₂) or subclass.

Unless otherwise indicated, when referring to antibodies (as broadlydefined herein), reference to antibody domains and/or amino acidpositions within antibodies, or fragments thereof, is in accordance withthe definition and assignment of amino acids to each domain in Kabat etal, SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5^(th) Ed. PublicHealth Service (National Institutes of Health, Bethesda, Md., 1987 and1991); incorporated herein by reference in its entirety. Amino acidsfrom the variable regions of the mature heavy and light chains ofimmunoglobulins are designated by the position of an amino acid in thechain. Kabat described numerous amino acid sequences for antibodies,identified an amino acid consensus sequence for each subgroup, andassigned a residue number to each amino acid. Kabat's numbering schemeis extendible to antibodies not included in his compendium by aligningthe antibody in question with one of the consensus sequences in Kabat byreference to conserved amino acids. This method for assigning residuenumbers has become standard in the field and readily identifies aminoacids at equivalent positions in different antibodies, includingchimeric or humanized variants. For example, an amino acid at position50 of a human antibody light chain occupies the equivalent position toan amino acid at position 50 of a mouse antibody light chain. Thus, asused herein in the context of humanized antibodies, a reference such as“at position 297 of the Fc region” refers to the amino acid position inan immunoglobulin chain, region of an a immunoglobulin chain, or regionof a polypeptide derived from an immunoglobulin chain, that correspondsto position 297 of the corresponding human immunoglobulin.

As used herein, the terms “B-cell malignancies” and “B-cell malignancy”refer to any B-cell lymphoproliferative disorder. B-cell malignanciesinclude tumors of B-cell origin. B-cell malignancies include, but arenot limited to, lymphomas, chronic lymphocytic leukemias, acutelymphoblastic leukemias, multiple myeloma, Hodgkin's and non-Hodgkin'sdisease, diffuse large B cell lymphoma, follicular lymphoma with areasof diffuse large B cell lymphoma, small lymphocytic lymphoma, mantlecell lymphoma, and diffuse small cleaved cell lymphoma.

As used herein, the term “derivative” in the context of a polypeptide orprotein, e.g. an antibody, refers to a polypeptide or protein thatcomprises an amino acid sequence which has been altered by theintroduction of amino acid residue substitutions, deletions oradditions. The term “derivative” as used herein also refers to apolypeptide or protein which has been modified, i.e., by the covalentattachment of any type of molecule to the antibody. For example, but notby way of limitation, a polypeptide or protein may be modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Aderivative a polypeptide or protein may be produced by chemicalmodifications using techniques known to those of skill in the art,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Further, aderivative a polypeptide or protein possesses a similar or identicalfunction as the a polypeptide or protein from which it was derived.

The term “derivative” as used herein in the context of a FcγRIIBantibody refers to an antibody that immunospecifically binds to aFcγRIIB polypeptide, or an antibody fragment that immunospecificallybinds to a FcγRIIB polypeptide, that has been altered by theintroduction of one or more amino acid residue substitutions, deletionsor additions (i.e., mutations) in one or more regions/domains of theantibody (e.g., CDRs, Fc region, hinge region, framework regions). Theantibody derivative may have substantially the same binding, betterbinding, or worse binding when compared to a non-derivative antibody. Inspecific embodiments, one, two, three, four, or five amino acid residuesof the CDR and/or Fc region have been substituted, deleted or added(i.e., mutated). The term “derivative” as used herein in conjunctionwith FcγRIIB also refers to an antibody that immunospecifically binds toa FcγRIIB polypeptide, or an antibody fragment that immunospecificallybinds to a FcγRIIB polypeptide which has been modified, i.e., by thecovalent attachment of any type of molecule to the polypeptide. Forexample, but not by way of limitation, an antibody, or antibody fragmentmay be modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. A derivative antibody, or antibody fragment may bemodified by chemical modifications using techniques known to those ofskill in the art, including, but not limited to, specific chemicalcleavage, acetylation, formulation, metabolic synthesis of tunicamycin,etc. Further, a derivative of an antibody, or antibody fragment maycontain one or more non-classical amino acids. In one embodiment, anantibody derivative possesses a similar or identical function as theparent antibody. In another embodiment, a derivative of an antibody, orantibody fragment has an altered activity when compared to an unalteredantibody. For example, a derivative antibody or fragment thereof canbind to its epitope more tightly or be more resistant to proteolysis.

As used herein, the terms “disorder” and “disease” are usedinterchangeably to refer to a condition in a subject. In particular, theterm “autoimmune disease” is used interchangeably with the term“autoimmune disorder” to refer to a condition in a subject characterizedby cellular, tissue and/or organ injury caused by an immunologicreaction of the subject to its own cells, tissues and/or organs. Theterm “inflammatory disease” is used interchangeably with the term“inflammatory disorder” to refer to a condition in a subjectcharacterized by inflammation, preferably chronic inflammation.Autoimmune disorders may or may not be associated with inflammation.Moreover, inflammation may or may not be caused by an autoimmunedisorder. Thus, certain disorders may be characterized as bothautoimmune and inflammatory disorders.

As used herein, the term “cancer” refers to a neoplasm or tumorresulting from abnormal uncontrolled growth of cells. As used herein,cancer explicitly includes, leukemias and lymphomas. The term “cancer”refers to a disease involving cells that have the potential tometastasize to distal sites and exhibit phenotypic traits that differfrom those of non-cancer cells, for example, formation of colonies in athree-dimensional substrate such as soft agar or the formation oftubular networks or weblike matrices in a three-dimensional basementmembrane or extracellular matrix preparation. Non-cancer cells do notform colonies in soft agar and form distinct sphere-like structures inthree-dimensional basement membrane or extracellular matrixpreparations. Cancer cells acquire a characteristic set of functionalcapabilities during their development, albeit through variousmechanisms. Such capabilities include evading apoptosis,self-sufficiency in growth signals, insensitivity to anti-growthsignals, tissue invasion/metastasis, limitless explicative potential,and sustained angiogenesis. The term “cancer cell” is meant to encompassboth pre-malignant and malignant cancer cells. In some embodiments,cancer refers to a benign tumor, which has remained localized. In otherembodiments, cancer refers to a malignant tumor, which has invaded anddestroyed neighboring body structures and spread to distant sites. Inyet other embodiments, the cancer is associated with a specific cancerantigen.

As used herein, the term “immunomodulatory agent” and variations thereofincluding, but not limited to, immunomodulatory agents, refer to anagent that modulates a host's immune system. In certain embodiments, animmunomodulatory agent is an immunosuppressant agent. In certain otherembodiments, an immunomodulatory agent is an immunostimulatory agent.Immunomodatory agents include, but are not limited to, small molecules,peptides, polypeptides, fusion proteins, antibodies, inorganicmolecules, mimetic agents, and organic molecules.

As used herein, the term “epitope” refers to a fragment of a polypeptideor protein having antigenic or immunogenic activity in an animal,preferably in a mammal, and most preferably in a human. An epitopehaving immunogenic activity is a fragment of a polypeptide or proteinthat elicits an antibody response in an animal. An epitope havingantigenic activity is a fragment of a polypeptide or protein to which anantibody immunospecifically binds as determined by any method well-knownto one of skill in the art, for example by immunoassays. Antigenicepitopes need not necessarily be immunogenic.

As used herein, the term “fragment” refers to a peptide or polypeptidecomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least contiguous 200 amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of anotherpolypeptide. In a specific embodiment, a fragment of a polypeptideretains at least one function of the polypeptide. Preferably, antibodyfragments are epitope binding fragments.

As used herein, the term “humanized antibody” refers to animmunoglobulin comprising a human framework region and one or more CDRsfrom a non-human (usually a mouse or rat) immunoglobulin. The non-humanimmunoglobulin providing the CDRs is called the “donor” and the humanimmunoglobulin providing the framework is called the “acceptor”.Constant regions need not be present, but if they are, they must besubstantially identical to human immunoglobulin constant regions, i.e.,at least about 85-90%, preferably about 95% or more identical. Hence,all parts of a humanized immunoglobulin, except possibly the CDRs, aresubstantially identical to corresponding parts of natural humanimmunoglobulin sequences. A “humanized antibody” is an antibodycomprising a humanized light chain and a humanized heavy chainimmunoglobulin. For example, a humanized antibody would not encompass atypical chimeric antibody, because, e.g., the entire variable region ofa chimeric antibody is non-human. The term “humanization” refers to theprocess of creating the humanized antibody. The resultant humanizedantibody is expected to bind to the same antigen as the donor antibodythat provides the CDRs. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ora non-human primate having the desired specificity, affinity, andcapacity. In some instances, Framework Region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable regions correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin thatimmunospecifically binds to a FcγRIIB polypeptide, that has been alteredby the introduction of amino acid residue substitutions, deletions oradditions (i.e., mutations). In some embodiments, a humanized antibodyis a derivative. Such a humanized antibody comprises amino acid residuesubstitutions, deletions or additions in one or more non-human CDRs. Thehumanized antibody derivative may have substantially the same binding,better binding, or worse binding when compared to a non-derivativehumanized antibody. In specific embodiments, one, two, three, four, orfive amino acid residues of the CDR have been substituted, deleted oradded (i.e., mutated). For further details in humanizing antibodies, seeEuropean Patent Nos. EP 239,400, EP 592,106, and EP 519,596;International Publication Nos. WO 91/09967 and WO 93/17105; U.S. Pat.Nos. 5,225,539, 5,530,101, 5,565,332, 5,585,089, 5,766,886, and6,407,213; and Padlan, 1991, Molecular Immunology 28(4/5):489-498;Studnicka et al., 1994, Protein Engineering 7(6):805-814; Roguska etal., 1994, PNAS 91:969-973; Tan et al., 2002, J. Immunol. 169:1119-25;Caldas et al., 2000, Protein Eng. 13:353-60; Morea et al., 2000, Methods20:267-79; Baca et al., 1997, J. Biol. Chem. 272:10678-84; Roguska etal., 1996, Protein Eng. 9:895-904; Couto et al., 1995, Cancer Res. 55(23 Supp):5973s-5977s; Couto et al., 1995, Cancer Res. 55:1717-22;Sandhu, 1994, Gene 150:409-10; Pedersen et al., 1994, J. Mol. Biol.235:959-73; Jones et al., 1986, Nature 321:522-525; Reichmann et al.,1988, Nature 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol.2:593-596; each of which is incorporated herein by reference in itsentirety.

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody which are responsible for antigen binding. Thehypervariable region comprises amino acid residues from a“Complementarity Determining Region” or “CDR” (i.e., residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variabledomain according to Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop”(i.e., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain; Chothia and Lesk, 1987, J. Mol. Biol.196:901-917; incorporated herein by reference in its entirety).“Framework Region” or “FR” residues are those variable domain residuesother than the hypervariable region residues as herein defined.

As used herein, the terms “single-chain Fv” or “scFv” refer to antibodyfragments comprise the VH and VL domains of antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the VH and VLdomains which enables the scFv to form the desired structure for antigenbinding. For a review of sFv, see Pluckthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315 (1994); incorporated herein byreference in its entirety. In specific embodiments, scFvs includebi-specific scFvs and humanized scFvs.

As used herein, the terms “nucleic acids” and “nucleotide sequences”include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g.,mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNAmolecules, and analogs of DNA or RNA molecules. Such analogs can begenerated using, for example, nucleotide analogs, which include, but arenot limited to, inosine or tritylated bases. Such analogs can alsocomprise DNA or RNA molecules comprising modified backbones that lendbeneficial attributes to the molecules such as, for example, nucleaseresistance or an increased ability to cross cellular membranes. Thenucleic acids or nucleotide sequences can be single-stranded,double-stranded, may contain both single-stranded and double-strandedportions, and may contain triple-stranded portions, but preferably isdouble-stranded DNA.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, a subject is preferably a mammal suchas a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and aprimate (e.g., monkey and human), most preferably a human.

As used herein, the terms “treat,” “treating” and “treatment” refer tothe eradication, reduction or amelioration of symptoms of a disease ordisorder related to the loss of regulation in the Fc receptor signalingpathway or to enhance the therapeutic efficacy of another therapy, e.g.,a therapeutic antibody, vaccine therapy or prophylaxis. In someembodiments, treatment refers to the eradication, removal, modification,or control of primary, regional, or metastatic cancer tissue thatresults from the administration of one or more therapeutic agents. Incertain embodiments, such terms refer to the minimizing or delaying thespread of cancer resulting from the administration of one or moretherapeutic agents to a subject with such a disease. In otherembodiments, such terms refer to elimination of disease causing cells.

As used herein, the phrase “side effects” encompasses unwanted andadverse effects of a prophylactic or therapeutic agent. Adverse effectsare always unwanted, but unwanted effects are not necessarily adverse.An adverse effect from a prophylactic or therapeutic agent might beharmful or uncomfortable or risky. Side effects from chemotherapyinclude, but are not limited to, gastrointestinal toxicity such as, butnot limited to, early and late-forming diarrhea and flatulence, nausea,vomiting, anorexia, leukopenia, anemia, neutropenia, asthenia, abdominalcramping, fever, pain, loss of body weight, dehydration, alopecia,dyspnea, insomnia, dizziness, mucositis, xerostomia, and kidney failure,as well as constipation, nerve and muscle effects, temporary orpermanent damage to kidneys and bladder, flu-like symptoms, fluidretention, and temporary or permanent infertility. Side effects fromradiation therapy include but are not limited to fatigue, dry mouth, andloss of appetite. Side effects from biological therapies/immunotherapiesinclude but are not limited to rashes or swellings at the site ofadministration, flu-like symptoms such as fever, chills and fatigue,digestive tract problems and allergic reactions. Side effects fromhormonal therapies include but are not limited to nausea, fertilityproblems, depression, loss of appetite, eye problems, headache, andweight fluctuation. Additional undesired effects typically experiencedby patients are numerous and known in the art, see, e.g., thePhysicians' Desk Reference (56^(th) ed., 2002), which is incorporatedherein by reference in its entirety.

As used herein, a “therapeutically effective amount” refers to thatamount of the therapeutic agent sufficient to treat or manage a diseaseor disorder associated with FcγRIIB and any disease related to the lossof regulation in the Fc receptor signaling pathway or to enhance thetherapeutic efficacy of another therapy, e.g., therapeutic antibody,vaccine therapy or prophylaxis, etc. A therapeutically effective amountmay refer to the amount of therapeutic agent sufficient to delay orminimize the onset of disease, e.g., delay or minimize the spread ofcancer. A therapeutically effective amount may also refer to the amountof the therapeutic agent that provides a therapeutic benefit in thetreatment or management of a disease. Further, a therapeuticallyeffective amount with respect to a therapeutic agent of the inventionmeans that amount of therapeutic agent alone, or in combination withother therapies, that provides a therapeutic benefit in the treatment ormanagement of a disease, e.g., sufficient to enhance the therapeuticefficacy of a therapeutic antibody sufficient to treat or manage adisease. Used in connection with an amount of FcγRIIB antibody of theinvention, the term can encompass an amount that improves overalltherapy, reduces or avoids unwanted effects, or enhances the therapeuticefficacy of or synergies with another therapeutic agent.

As used herein, the terms “prophylactic agent” and “prophylactic agents”refer to any agent(s) which can be used in the prevention of a disorder,or prevention of recurrence or spread of a disorder. A prophylacticallyeffective amount may refer to the amount of prophylactic agentsufficient to prevent the recurrence or spread of hyperproliferativedisease, particularly cancer, or the occurrence of such in a patient,including but not limited to those predisposed to hyperproliferativedisease, for example those genetically predisposed to cancer orpreviously exposed to carcinogens. A prophylactically effective amountmay also refer to the amount of the prophylactic agent that provides aprophylactic benefit in the prevention of disease. Further, aprophylactically effective amount with respect to a prophylactic agentof the invention means that amount of prophylactic agent alone, or incombination with other agents, that provides a prophylactic benefit inthe prevention of disease. Used in connection with an amount of anFcγRIIB antibody of the invention, the term can encompass an amount thatimproves overall prophylaxis or enhances the prophylactic efficacy of orsynergies with another prophylactic agent, such as but not limited to atherapeutic antibody. In certain embodiments, the term “prophylacticagent” refers to an agonistic FcγRIIB-specific antibody. In otherembodiments, the term “prophylactic agent” refers to an antagonisticFcγRIIB-specific antibody. In certain other embodiments, the term“prophylactic agent” refers to cancer chemotherapeutics, radiationtherapy, hormonal therapy, biological therapy (e.g., immunotherapy),and/or FcγRIIB antibodies of the invention. In other embodiments, morethan one prophylactic agent may be administered in combination.

As used herein, the terms “manage,” “managing” and “management” refer tothe beneficial effects that a subject derives from administration of aprophylactic or therapeutic agent, which does not result in a cure ofthe disease. In certain embodiments, a subject is administered one ormore prophylactic or therapeutic agents to “manage” a disease so as toprevent the progression or worsening of the disease.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the prevention of the occurrence and/or recurrence or onset of one ormore symptoms of a disorder in a subject resulting from theadministration of a prophylactic or therapeutic agent.

As used herein, the term “in combination” refers to the use of more thanone prophylactic and/or therapeutic agents. The use of the term “incombination” does not restrict the order in which prophylactic and/ortherapeutic agents are administered to a subject with a disorder, e.g.,hyperproliferative cell disorder, especially cancer. A firstprophylactic or therapeutic agent can be administered prior to (e.g., 1minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours,4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeksbefore), concomitantly with, or subsequent to (e.g., 1 minute, 5minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after)the administration of a second prophylactic or therapeutic agent to asubject which had, has, or is susceptible to a disorder. Theprophylactic or therapeutic agents are administered to a subject in asequence and within a time interval such that the agent of the inventioncan act together with the other agent to provide an increased benefitthan if they were administered otherwise. Any additional prophylactic ortherapeutic agent can be administered in any order with the otheradditional prophylactic or therapeutic agents.

5. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B. A. AMINO ACID ALIGNMENTS. The alignment of the aminoacid sequences of mouse 2B6 VH (SEQ ID NO:28), humanized 2B6 VH-1 (SEQID NO:24), VH1-18 (SEQ ID NOS:4-6; SEQ ID NOS:71-72) and human JH6 (SEQID NOS:4-7, SEQ ID NOS:71-73) is shown in FIG. 1A. B. AMINO ACIDALIGNMENTS. This figure shows the alignment of amino acid sequences ofmurine 2B6 VL (SEQ ID NO:26), human 2B6 VL-1 (SEQ ID NO:18), human 2B6VL-2 (SEQ ID NO:20); human 2B6 VL-3 (SEQ ID NO:22), and human VK-A26(SEQ ID NOS:13-15; SEQ ID NOS:74-76) and JK4 (SEQ ID NOS:13-16; SEQ IDNOS:74-76).

FIGS. 2A and 2B A. AMINO ACID ALIGNMENTS. The alignment of the aminoacid sequences of humanized 2B6 VH-1 (SEQ ID NO:24) and humanized 2B6VH3 (SEQ ID NO:68) is shown in FIG. 2A. B. AMINO ACID ALIGNMENTS. Thisfigure shows the alignment of amino acid sequences of humanized 2B6 VL-1(SEQ ID NO:18), 2B6 VL-2 (SEQ ID NO:20), 2B6 VL-3 (SEQ ID NO:22), and2B6 VL-5 (SEQ ID NO:62). For FIGS. 2A and 2B, CDRs are indicated byunderlining, and differences in amino acid sequence are indicated bybold lettering.

FIG. 3. BINDING OF hu2B6HC/ch2B6LC mAb AND ch2B6 mAb TO FcγRIIB. Bindingto dimeric soluble FcγRIIB-Fc was determined by ELISA. hu2B6HC/ch2B6LCmonoclonal antibody bound to the receptor with similar affinity to thech2B6 monoclonal antibody.

FIG. 4. BINDING OF ch2B6 mAb, h2B6 (v3.5) mAb and h2B6 (v1.1) mAb toFcγRIIB. Binding to dimeric soluble FcγRIIB-Fc was determined by ELISA.A hu2B6 (v3.5) monoclonal antibody bound to the receptor with similaraffinity to the ch2B6 monoclonal antibody. Antibody

FIG. 5. BINDING OF hu2B6LC/ch2B6HC mAB, ch2B6LC/hu2B6HC, AND ch2B6 mAbTO FcγRIIB. Binding to dimeric soluble FcγRIIB-Fc was determined byELISA. hu2B6HC/ch2B6LC mAb and ch2B6HC/hu2B6LC mAB bound to the receptorwith similar affinity to the ch2B6 mAb.

FIG. 6. BINDING OF hu2B6 VARIANTS TO FcγRIIB. Binding of Hu2B6N50Y;Hu2B6N50Y, V51A; Ch2B6, and Hu2B6 to dimeric soluble FcγRIIb-Fc wasdetermined by ELISA. All of the mAbs bound to the receptor with similaraffinity.

FIG. 7. BINDING OF hu2B6 VARIANTS TO FcγRIIA. Binding of Hu2B6N50Y;Hu2B6N50Y, V51A; Ch2B6, and Hu2B6 to dimeric soluble FcγRIIa-Fc wasdetermined by ELISA. The humanized 2B6 mAbs selectively bind to CD32B.All of the solid data points fall on top of each other and are onlydisplayed as a solid square.

FIGS. 8 A and 8 B. ESTIMATED TUMOR WEIGHT IN MICE TREATED WITH WILD-TYPEOR Fc MUTANT h2B6. Balb/c nude mice were inoculated subcutaneously withDaudi cells and administered 25 μg, 2.5 μg or 0.25 μg weekly doses ofeither wild-type h2B6 (A) or a variant h2B6 comprising an Fc domain witha leucine at position 243, a proline at position 292, a leucine atposition 300, an isoleucine at position 305, and a leucine at position396; (B). Mice administered buffer alone were used as control. Tumorweight was calculated based on the estimated volume of the subcutaneoustumor according to the formula (width X length)/2.

FIGS. 9 A and 9 B. SURVIVAL IN TUMOR BEARING MICE TREATED WITH WILD-TYPEOR Fc MUTANT h2B6. Nude mice were inoculated with Daudi cells andadministered 25 μg, 2.5 μg or 0.25 μg weekly doses of either wild-typeh2B6 (A) or a variant h2B6 comprising an Fc domain with 243L, 292P,300L, 305I, and 396L; (B). Mice administered buffer alone were used ascontrol.

6. DESCRIPTION OF THE PREFERRED EMBODIMENTS

6.1 FcγRIIB-Specific Antibodies

The present invention encompasses humanized antibodies (preferablyhumanized monoclonal antibodies) or fragments thereof that specificallybind FcγRIIB, preferably human FcγRIIB, more preferably native humanFcγRIIB with a greater affinity than said antibodies or fragmentsthereof bind FcγRIIA, preferably human FcγRIIA, more preferably nativehuman FcγRIIA. Preferably, the humanized antibodies of the inventionbind the extracellular domain of native human FcγRIIB. In certainembodiments, the humanized antibodies or fragments thereof bind toFcγRIIB with an affinity greater than two-fold, four fold, 6 fold, 10fold, 20 fold, 50 fold, 100 fold, 1000 fold, 10⁴ fold, 10⁵ fold, 10⁶fold, 10⁷ fold, or 10⁸ fold than said antibodies or fragments thereofbind FcγRIIA. In one particular embodiment, the humanized antibody ofthe invention is derived from a mouse monoclonal antibody produced byclone 2B6 or 3H7, having ATCC accession numbers PTA-4591 and PTA-4592,respectively. In another embodiment, the humanized antibody of theinvention is derived from a mouse monoclonal antibody produced by clone1D5, 2E1, 2H9, 2D11, or 1F2, having ATCC Accession numbers, PTA-5958,PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively. Hybridomasproducing antibodies 2B6 and 3H7 have been deposited with the AmericanType Culture Collection (10801 University Blvd., Manassas, Va.20110-2209) on Aug. 13, 2002 under the provisions of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedures, and assigned accession numbersPTA-4591 (for hybridoma producing 2B6) and PTA-4592 (for hybridomaproducing 3H7), respectively, and are incorporated herein by reference.Hybridomas producing 1D5, 2E1, 2H9, 2D11, and 1F2 were deposited underthe provisions of the Budapest Treaty with the American Type CultureCollection (10801 University Blvd., Manassas, Va. 20110-2209) on May 7,2004, and assigned accession numbers PTA-5958, PTA-5961, PTA-5962,PTA-5960, and PTA-5959, respectively and are incorporated herein byreference.

In yet other embodiments, the invention encompasses humanized FcγRIIBantibodies that bind exclusively to FcγRIIB and have no affinity forFcγRIIA using standard methods known in the art and disclosed herein.

In a specific embodiment, the invention encompasses a humanized antibodycomprising the CDRs of 2B6 or of 3H7. In particular, the inventionencompasses a humanized antibody with the heavy chain variable domainhaving the amino acid sequence of SEQ ID NO: 24, SEQ ID NO:60 or SEQ IDNO:68 and the light chain variable domain having the amino acid sequenceof SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:62. In aspecific embodiment, the invention encompasses a humanized antibody withthe heavy chain variable domain having the amino acid sequence of SEQ IDNO: 37 and the light chain variable domain having the amino acidsequence of SEQ ID NO: 46. In a preferred embodiment, the inventionencompasses a humanized antibody with the heavy chain variable domainhaving the amino acid sequence of SEQ ID NO:68 and the light chainvariable domain having the amino acid sequence of SEQ ID NO:62. In yetanother preferred embodiment, the humanized antibodies of the inventionfurther do not bind Fc activation receptors, e.g., FcγIIIA, FcγIIIB,etc. In one embodiment, the humanized FcγRIIB-specific antibody inaccordance with the invention is not derived from the monoclonalantibody designated KB61, as disclosed in Pulford et al., 1986(Immunology, 57: 71-76) or the monoclonal antibody designated MAbII8D2as disclosed in Weinrich et al., 1996, (Hybridoma, 15(2):109-6); each ofwhich reference is incorporated herein by reference in its entirety. Ina specific embodiment, the FcγRIIB-specific antibody of the inventiondoes not bind to the same epitope and/or does not compete with bindingwith the monoclonal antibody KB61 or II8D2. Preferably, the humanizedFcγRIIB-specific antibodies of the invention do not bind the amino acidsequence SDPNFSI corresponding to positions 135-141 of FcγRIIb2 isoform.

The constant domains of the humanized antibodies of the invention may beselected with respect to the proposed function of the antibody, inparticular with regard to the effector function which may be required.In some embodiments, the constant domains of the humanized antibodies ofthe invention are human IgA, IgE, IgG or IgM domains. In a specificembodiment, human IgG constant domains, especially of the IgG1 and IgG3isotypes are used, especially when the humanized antibodies of theinvention are intended for therapeutic uses and antibody effectorfunctions are needed. In alternative embodiments, IgG2 and IgG4 isotypesare used when the humanized antibody of the invention is intended fortherapeutic purposes and antibody effector function is not required. Inother embodiments, the invention encompasses humanized antibodiescomprising one or more amino acid modifications in the Fc region such asthose disclosed in International Publication Nos. WO 04/063351, WO04/029207, WO 04/029092, WO 04/028564, WO 99/58572, WO 99/51642, WO98/23289, WO 89/07142, WO 88/07089; U.S. Patent Application PublicationNos. 2005/0037000; and 2005/0064514 and U.S. Pat. Nos. 5,843,597 and5,642,821; U.S. patent application Ser. Nos. 10/902,588 (filed Jul. 28,2004) and 11/271,140 (filed Nov. 10, 2005); U.S. Provisional ApplicationNos. 60/707,419; and 60/781,564 filed on Aug. 10, 2005, and Mar. 10,2006, respectively; and U.S. Pat. Nos. 5,624,821 and 5,648,260 andEuropean Patent No. EP 0 307 434; all of which are incorporated hereinby reference in their entireties. In a specific embodiment, the aminoacid modification of the Fc regions of the humanized antibodies of theinvention relative to a wild-type Fc region comprise a substitution atposition 243, 292, 300, 305 and 396. In a preferred embodiment, theamino acid modification of the Fc regions of the humanized antibodies ofthe invention relative to a wild-type Fc region comprise a substitutionat position 243 with leucine, at position 292 with proline, at position300 with leucine, at position 305 with isoleucine and at position 396with leucine.

Preferably, the humanized antibodies of the invention bind theextracellular domain of native human FcγRIIB that is endogenouslyexpressed on the surface of a cell. The humanized anti-FcγRIIBantibodies of the invention may have a heavy chain variable regioncomprising the amino acid sequence of a CDR1 (e.g., SEQ ID NO:1, SEQ IDNO:29, an amino acid sequence corresponding to amino acids 31-35 as setforth in SEQ ID NO:60, or an amino acid sequence corresponding to aminoacids 31-35 as set forth in SEQ ID NO:68) and/or a CDR2 (e.g., SEQ IDNO:2, SEQ ID NO:30, an amino acid sequence corresponding to amino acids50-66 as set forth in SEQ ID NO:60, or an amino acid sequencecorresponding to amino acids 50-66 as set forth in SEQ ID NO:68) and/ora CDR3 (e.g., SEQ ID NO:3, SEQ ID NO:31, an amino acid sequencecorresponding to amino acids 99-110 as set forth in SEQ ID NO:60, or anamino acid sequence corresponding to amino acids 99-110 as set forth inSEQ ID NO:68) and/or a light chain variable region comprising the aminoacid sequence of a CDR1 (e.g., SEQ ID NO:8, SEQ ID NO:38, or an aminoacid sequence corresponding to amino acids 24-34 as set forth in SEQ IDNO:62) and/or a CDR2 (e.g., SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:39, or an amino acid sequence corresponding to amino acids 50-56as set forth in SEQ ID NO:62) and/or a CDR3 (e.g., SEQ ID NO:12, SEQ IDNO:40, or an amino acid sequence corresponding to amino acids 90-98 asset forth in SEQ ID NO:62).

In a specific embodiment, the invention provides a humanized 2B6antibody, wherein the VH region consists of the FR segments from thehuman germline VH segment VH1-18 (Matsuda et al., 1998, J. Exp. Med.188:2151062) and JH6 (Ravetch et al., 1981, Cell 27(3 Pt. 2): 583-91)(each of which reference is incorporated herein by reference in itsentirety), and one or more CDR regions of the 2B6 VH, having the aminoacid sequence of SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3. Inspecific embodiments, the 2B6 VH has the amino acid sequence of SEQ IDNO. 24, SEQ ID NO:60, or SEQ ID NO:68. In another specific embodiment,the humanized 2B6 antibody further comprises a VL region, which consistsof the FR segments of the human germline VL segment VK-A26(Lautner-Rieske et al., 1992, Eur. J. Immunol. 22:1023-1029) and JK4(Hieter et al., 1982, J. Biol. Chem. 257:1516-22) (each of whichreference is incorporated herein by reference in its entirety), and oneor more CDR regions of 2B6 VL, having the amino acid sequence of SEQ IDNO: 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12. Inone embodiment, the 2B6 VL has the amino acid sequence of SEQ ID NO. 18;SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO:62. In a preferredembodiment, the 2B6 antibody of the invention comprises a VL comprisingthe amino acid sequence SEQ ID NO:62 and a VH comprising the amino acidsequence SEQ ID NO:68.

In another specific embodiment, the invention provides a humanized 3H7antibody, wherein the VH region consists of the FR segments from a humangermline VH segment and the CDR regions of the 3H7 VH, having the aminoacid sequence of SEQ ID NO. 37. In another specific embodiment, thehumanized 3H7 antibody further comprises a VL regions, which consists ofthe FR segments of a human germline VL segment and the CDR regions of3H7 VL, having the amino acid sequence of SEQ ID NO. 46.

In particular, the invention provides a humanized antibody thatimmunospecifically binds to extracellular domain of native humanFcγRIIB, said antibody comprising (or alternatively, consisting of) CDRsequences of 2B6 or 3H7, in any of the following combinations: a VH CDR1and a VL CDR1; a VH CDR1 and a VL CDR2; a VH CDR1 and a VL CDR3; a VHCDR2 and a VL CDR1; VH CDR2 and VL CDR2; a VH CDR2 and a VL CDR3; a VHCDR3 and a VH CDR1; a VH CDR3 and a VL CDR2; a VH CDR3 and a VL CDR3; aVH1 CDR1, a VH CDR2 and a VL CDR1; a VH CDR1, a VH CDR2 and a VL CDR2; aVH CDR1, a VH CDR2 and a VL CDR3; a VH CDR2, a VH CDR3 and a VL CDR1, aVH CDR2, a VH CDR3 and a VL CDR2; a VH CDR2, a VH CDR2 and a VL CDR3; aVH CDR1, a VL CDR1 and a VL CDR2; a VH CDR1, a VL CDR1 and a VL CDR3; aVH CDR2, a VL CDR1 and a VL CDR2; a VH CDR2, a VL CDR1 and a VL CDR3; aVH CDR3, a VL CDR1 and a VL CDR2; a VH CDR3, a VL CDR1 and a VL CDR3; aVH CDR1, a VH CDR2, a VH CDR3 and a VL CDR1; a VH CDR1, a VH CDR2, a VHCDR3 and a VL CDR2; a VH CDR1, a VH CDR2, a VH CDR3 and a VL CDR3; a VHCDR1, a VH CDR2, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR2, a VLCDR1 and a VL CDR3; a VH CDR1, a VH CDR3, a VL CDR1 and a VL CDR2; a VHCDR1, a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR2, a VH CDR3, a VLCDR1 and a VL CDR2; a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR3; a VHCDR2, a VH CDR3, a VL CDR2 and a VL CDR3; a VH CDR1, a VH CDR2, a VHCDR3, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR2, a VH CDR3, a VLCDR1 and a VL CDR3; a VH CDR1, a VH CDR2, a VL CDR1, a VL CDR2, and a VLCDR3; a VH CDR1, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3; a VHCDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3; or any combinationthereof of the VH CDRs and VL CDRs disclosed herein.

The present invention provides humanized antibody molecules specific forFcγRIIB in which one or more regions of one or more CDRs of the heavyand/or light chain variable regions of a human antibody (the recipientantibody) have been substituted by analogous parts of one or more CDRsof a donor monoclonal antibody which specifically binds FcγRIIB, with agreater affinity than FcγRIIA, e.g., a monoclonal antibody produced byclone 2B6 or 3H7, having ATCC accession numbers PTA-4591, and PTA-4592,respectively, or a monoclonal antibody produced by clone 1D5, 2E1, 2H9,2D11, or 1F2, having ATCC Accession numbers, PTA-5958, PTA-5961,PTA-5962, PTA-5960, and PTA-5959, respectively. In other embodiments,the humanized antibodies of the invention bind to the same epitope as2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2. In a most preferred embodiment,the humanized antibody specifically binds to the same epitope as thedonor murine antibody. It will be appreciated by one skilled in the artthat the invention encompasses CDR grafting of antibodies in general.Thus, the donor and acceptor antibodies may be derived from animals ofthe same species and even same antibody class or sub-class. Moreusually, however, the donor and acceptor antibodies are derived fromanimals of different species. Typically the donor antibody is anon-human antibody, such as a rodent MAb, and the acceptor antibody is ahuman antibody.

In some embodiments, at least one CDR from the donor antibody is graftedonto the human antibody. In other embodiments, at least two andpreferably all three CDRs of each of the heavy and/or light chainvariable regions are grafted onto the human antibody. The CDRs maycomprise the Kabat CDRs, the structural loop CDRs or a combinationthereof. In some embodiments, the invention encompasses a humanizedFcγRIIB antibody comprising at least one CDR grafted heavy chain and atleast one CDR-grafted light chain.

In a preferred embodiment, the CDR regions of the humanized FcγRIIBspecific antibody are derived from a murine antibody specific forFcγRIIB. In some embodiments, the humanized antibodies described hereincomprise alterations, including but not limited to amino acid deletions,insertions, modifications, of the acceptor antibody, i.e., human, heavyand/or light chain variable domain framework regions that are necessaryfor retaining binding specificity of the donor monoclonal antibody. Insome embodiments, the framework regions of the humanized antibodiesdescribed herein does not necessarily consist of the precise amino acidsequence of the framework region of a natural occurring human antibodyvariable region, but contains various alterations, including but notlimited to amino acid deletions, insertions, modifications that alterthe property of the humanized antibody, for example, improve the bindingproperties of a humanized antibody region that is specific for the sametarget as the murine FcγRIIB specific antibody. In most preferredembodiments, a minimal number of alterations are made to the frameworkregion in order to avoid large-scale introductions of non-humanframework residues and to ensure minimal immunogenicity of the humanizedantibody in humans. The donor monoclonal antibody of the presentinvention is preferably a monoclonal antibody produced by clones 2B6 and3H7 (having ATCC accession numbers PTA-4591, and PTA-4592, respectively)which bind FcγRIIB or a monoclonal antibody produced by clones 1D5, 2E1,2H9, 2D11, or 1F2 (having ATCC Accession numbers, PTA-5958, PTA-5961,PTA-5962, PTA-5960, and PTA-5959, respectively).

In a specific embodiment, the invention encompasses a CDR-graftedantibody which specifically binds FcγRIIB with a greater affinity thansaid antibody binds FcγRIIA, wherein the CDR-grafted antibody comprisesa heavy chain variable region domain comprising framework residues ofthe recipient antibody and residues from the donor monoclonal antibody,which specifically binds FcγRIIB with a greater affinity than saidantibody binds FcγRIIA, e.g., monoclonal antibody produced from clones2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2. In a specific embodiment, theheavy chain variable domain of the antibodies of the invention comprisethe amino acid sequence SEQ ID NO:60. In another specific embodiment,the heavy chain variable domain of the antibodies of the inventioncomprise the amino acid sequence SEQ ID NO:68. In other embodiments, theinvention encompasses a CDR-grafted antibody which specifically bindsFcγRIIB with a greater affinity than said antibody binds FcγRIIA,wherein the CDR-grafted antibody comprises a light chain variable regiondomain comprising framework residues of the recipient antibody andresidues from the donor monoclonal antibody, which specifically bindsFcγRIIB with a greater affinity than said antibody binds FcγRIIA, e.g.,monoclonal antibody produced from clones 2B6, 3H7, 1D5, 2E1, 2H9, 2D11,or 1F2. In a specific embodiment, the light chain variable domain of theantibodies of the invention comprise the amino acid sequence SEQ IDNO:62.

A humanized FcγRIIB specific antibody of the invention may comprisesubstantially all of at least one, and typically two, variable domainsin which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin (i.e., donor antibody) and all orsubstantially all of the framework regions are those of a humanimmunoglobulin consensus sequence. Preferably, a humanized antibody ofthe invention also comprises at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Theconstant domains of the humanized antibodies of the invention may beselected with respect to the proposed function of the antibody, inparticular the effector function which may be required. In someembodiments, the constant domains of the humanized antibodies of theinvention are human IgA, IgE, IgG or IgM domains. In a specificembodiment, human IgG constant domains, especially of the IgG1 and IgG3isotypes are used, when the humanized antibodies of the invention isintended for therapeutic uses and antibody effector functions areneeded. In alternative embodiments, IgG2 and IgG4 isotypes are used whenthe humanized antibody of the invention is intended for therapeuticpurposes and antibody effector function is not required. The inventionencompasses Fc constant domains comprising one or more amino acidmodifications which alter antibody effector functions such as thosedisclosed in U.S. Patent Application Publication Nos. 2005/0037000 and2005/0064514; U.S. Provisional Application Nos. 60/439,498; 60/456,041;and 60/514,549 filed on Jan. 9, 2003; Mar. 19, 2003, and Oct. 23, 2003respectively; all of which are incorporated herein by reference in theirentireties. In a specific embodiment, the antibody of the inventioncomprises an Fc domain having a laucine at position 243, a praline atposition 292, a leucine at position 300, an isoleucine at position 305,and a leucine at position 396. In other embodiment, the antibody of theinvention comprises amino acid modification(s) of the Fc region relativeto a wild-type Fc region, which modification comprise a substitution atposition 243, 292, 300, 305 and 396, preferably a substitution atposition 243 with leucine, at position 292 with proline, at position 300with leucine, at position 305 with isoleucine and at position 396 withleucine.

In some embodiments, the humanized antibody of the invention containsboth the light chain as well as at least the variable domain of a heavychain. In other embodiments, the humanized antibody of the invention mayfurther comprise one or more of the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. The humanized antibody can be selected fromany class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, andany isotype, including IgG₁, IgG₂, IgG₃ and IgG₄. In some embodiments,the constant domain is a complement fixing constant domain where it isdesired that the humanized antibody exhibit cytotoxic activity, and theclass is typically IgG₁. In other embodiments, where such cytotoxicactivity is not desirable, the constant domain may be of the IgG₂ class.The humanized antibody of the invention may comprise sequences from morethan one class or isotype, and selecting particular constant domains tooptimize desired effector functions is within the ordinary skill in theart.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor CDR orthe consensus framework may be mutagenized by substitution, insertion ordeletion of at least one residue so that the CDR or framework residue atthat site does not correspond to either the consensus or the donorantibody. Such mutations, however, are preferably not extensive.Usually, at least 75% of the humanized antibody residues will correspondto those of the parental framework region (FR) and CDR sequences, moreoften 90%, and most preferably greater than 95%. Humanized antibodiescan be produced using variety of techniques known in the art, including,but not limited to, CDR-grafting (European Patent No. EP 239,400;International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539,5,530,101, and 5,585,089), veneering or resurfacing (European PatentNos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology28(4/5):489-498; Studnicka et al., 1994, Protein Engineering7(6):805-814; and Roguska et al., 1994, Proc. Natl. Acad. Sci.91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and techniquesdisclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886, 5,585,089,International Publication No. WO 9317105, Tan et al., 2002, J. Immunol.169:1119-25, Caldas et al., 2000, Protein Eng. 13:353-60, Morea et al.,2000, Methods 20:267-79, Baca et al., 1997, J. Biol. Chem. 272:10678-84,Roguska et al., 1996, Protein Eng. 9:895-904, Couto et al., 1995, CancerRes. 55 (23 Supp):5973s-5977s, Couto et al., 1995, Cancer Res.55:1717-22, Sandhu, 1994, Gene 150:409-10, Pedersen et al., 1994, J.Mol. Biol. 235:959-73, Jones et al., 1986, Nature 321:522-525, Riechmannet al., 1988, Nature 332:323, and Presta, 1992, Curr. Op. Struct. Biol.2:593-596; each of which is incorporated herein by reference in itsentirety. Often, framework residues in the framework regions will besubstituted with the corresponding residue from the CDR donor antibodyto alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; U.S.Publication Nos. 2004/0049014 and 2003/0229208; U.S. Pat. Nos.6,350,861; 6,180,370; 5,693,762; 5,693,761; 5,585,089; and 5,530,101 andRiechmann et al., 1988, Nature 332:323, all of which are incorporatedherein by reference in their entireties.)

In a particular embodiment, the humanized antibodies of the invention,or fragments thereof, agonize at least one activity of FcγRIIB. In oneembodiment of the invention, said activity is inhibition of B cellreceptor-mediated signaling. In another embodiment, the humanizedagonistic antibodies of the invention inhibit activation of B cells, Bcell proliferation, antibody production, intracellular calcium influx ofB cells, cell cycle progression, or activity of one or more downstreamsignaling molecules in the FcγRIIB signal transduction pathway. In yetanother embodiment, the humanized agonistic antibodies of the inventionenhance phosphorylation of FcγRIIB or SHIP recruitment. In a furtherembodiment of the invention, the humanized agonistic antibodies inhibitMAP kinase activity or Akt recruitment in the B cell receptor-mediatedsignaling pathway. In another embodiment, the humanized agonisticantibodies of the invention agonize FcγRIIB-mediated inhibition of FcεRIsignaling. In a particular embodiment, said humanized antibodies inhibitFcεRI-induced mast cell activation, calcium mobilization, degranulation,cytokine production, or serotonin release. In another embodiment, thehumanized agonistic antibodies of the invention stimulatephosphorylation of FcγRIIB, stimulate recruitment of SHIP, stimulateSHIP phosphorylation and its association with Shc, or inhibit activationof MAP kinase family members (e.g., Erk1, Erk2, JNK, p38, etc.). In yetanother embodiment, the humanized agonistic antibodies of the inventionenhance tyrosine phosphorylation of p62dok and its association with SHIPand rasGAP. In another embodiment, the humanized agonistic antibodies ofthe invention inhibit FcγR-mediated phagocytosis in monocytes ormacrophages.

In another embodiment, the humanized antibodies of the invention, orfragments thereof, antagonize at least one activity of FcγRIIB. In oneembodiment, said activity is activation of B cell receptor-mediatedsignaling. In a particular embodiment, the humanized antagonisticantibodies of the invention enhance B cell activity, B cellproliferation, antibody production, intracellular calcium influx, oractivity of one or more downstream signaling molecules in the FcγRIIBsignal transduction pathway. In yet another particular embodiment, thehumanized antagonistic antibodies of the invention decreasephosphorylation of FcγRIIB or SHIP recruitment. In a further embodimentof the invention, the humanized antagonistic antibodies enhance MAPkinase activity or Akt recruitment in the B cell receptor mediatedsignaling pathway. In another embodiment, the humanized antagonisticantibodies of the invention antagonize FcγRIIB-mediated inhibition ofFcεRI signaling. In a particular embodiment, the humanized antagonisticantibodies of the invention enhance FcεRI-induced mast cell activation,calcium mobilization, degranulation, cytokine production, or serotoninrelease. In another embodiment, the humanized antagonistic antibodies ofthe invention inhibit phosphorylation of FcγRIIB, inhibit recruitment ofSHIP, inhibit SHIP phosphorylation and its association with Shc, enhanceactivation of MAP kinase family members (e.g., Erk1, Erk2, JINK, p38,etc.). In yet another embodiment, the humanized antagonistic antibodiesof the invention inhibit tyrosine phosphorylation of p62dok and itsassociation with SHIP and rasGAP. In another embodiment, the humanizedantagonistic antibodies of the invention enhance FcγR-mediatedphagocytosis in monocytes or macrophages. In another embodiment, thehumanized antagonistic antibodies of the invention prevent phagocytosis,clearance of opsonized particles by splenic macrophages.

Antibodies of the invention include, but are not limited to, monoclonalantibodies, synthetic antibodies, recombinantly produced antibodies,multispecific antibodies, human antibodies, chimeric antibodies,camelized antibodies, single-chain Fvs (scFv), single chain antibodies,Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFv),intrabodies, and epitope-binding fragments of any of the above. Inparticular, antibodies used in the methods of the present inventioninclude immunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site that immunospecifically binds to FcγRIIB with greateraffinity than said immunoglobulin molecule binds FcγRIIA and/orimmunospecifically binds FcγRIIB and blocks the Fc binding domain ofFcγRIIB. Antibody analogs may also include FcγRIIB-specific T-cellreceptors, for example, chimeric T-cell receptors (see, e.g., U.S.Patent Application Publication No. 2004/0043401), a single-chain T-cellreceptor linked to a single-chain antibody (see, e.g., U.S. Pat. No.6,534,633), and protein scaffolds (see, e.g., U.S. Pat. No. 6,818,418)(each of which reference is incorporated herein by reference in itsentirety). In certain embodiments, an antibody analog of the inventionis not a monoclonal antibody.

The humanized antibodies used in the methods of the invention may befrom any animal origin including birds and mammals (e.g., human,non-human primate, murine, donkey, sheep, rabbit, goat, guinea pig,camel, horse, or chicken). Preferably, the antibodies are human orhumanized monoclonal antibodies. As used herein, “human” antibodiesinclude antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries or libraries of synthetic human immunoglobulin codingsequences or from mice that express antibodies from human genes.

The humanized antibodies used in the methods of the present inventionmay be monospecific, bispecific, trispecific or of greatermultispecificity. Multispecific antibodies may immunospecifically bindto different epitopes of FcγRIIB or immunospecifically bind to both anepitope of FcγRIIB as well a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g.,International Publication Nos. WO 93/17715, WO 92/08802, WO 91/00360,and WO 92/05793; Tutt, et al., 1991, J. Immunol. 147:60-69; U.S. Pat.Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and 5,601,819; andKostelny et al., 1992, J. Immunol. 148:1547-1553; Todorovska et al.,2001 Journal of Immunological Methods, 248:47-66; each of which isincorporated herein by reference in its entirety. In particularembodiments, the humanized antibodies of the invention are multispecificwith specificities for FcγRIIB and for a cancer antigen or any othercell surface marker specific for a cell (e.g., an immune cell such as aT-cell or B-cell) designed to be killed, e.g., in treating or preventinga particular disease or disorder, or for other Fc receptors, e.g.,FcγRIIIA, FcγRIIIB, etc.

In a specific embodiment, an antibody used in the methods of the presentinvention is an antibody or an antigen-binding fragment thereof (e.g.,comprising one or more complementarily determining regions (CDRs),preferably all 6 CDRs) of the antibody produced by clone 2B6, 3H7, 1D5,2E1, 2H9, 2D11, or 1F2, with ATCC accession numbers PTA-4591, PTA-4592,PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively(e.g., the heavy chain CDR3). In another embodiment, an antibody used inthe methods of the present invention binds to the same epitope as themouse monoclonal antibody produced from clone 2B6, 3H7, 1D5, 2E1, 2H9,2D11, or 1F2, with ATCC accession numbers PTA-4591, PTA-4592, PTA-5958,PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively and/or competeswith the mouse monoclonal antibody produced from clone 2B6, 3H7, 1D5,2E1, 2H9, 2D11, or 1F2, with ATCC accession numbers PTA-4591, PTA-4592,PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively asdetermined, e.g., in an ELISA assay or other appropriate competitiveimmunoassay, and also binds FcγRIIB with a greater affinity than saidantibody or a fragment thereof binds FcγRIIA.

The humanized antibodies used in the methods of the invention includederivatives that are modified, i.e., by the covalent attachment of anytype of molecule to the antibody such that covalent attachment. Forexample, but not by way of limitation, the antibody derivatives includeantibodies that have been modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. Any of numerous chemical modifications maybe carried out by known techniques, including, but not limited to,specific chemical cleavage, acetylation, formylation, metabolicsynthesis of tunicamycin, etc. Additionally, the derivative may containone or more non-classical amino acids.

For some uses, including in vivo use of humanized antibodies in humansand in vitro detection assays, it may be preferable to use human,chimeric or humanized antibodies. Completely human antibodies areparticularly desirable for therapeutic treatment of human subjects.Human antibodies can be made by a variety of methods known in the artincluding phage display methods described above using antibody librariesderived from human immunoglobulin sequences. See also U.S. Pat. Nos.4,444,887 and 4,716,111; and International Publication Nos. WO 98/46645,WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO91/10741; each of which is incorporated herein by reference in itsentirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of theJ_(H) region prevents endogenous antibody production. The modifiedembryonic stem cells are expanded and microinjected into blastocysts toproduce chimeric mice. The chimeric mice are then bred to producehomozygous offspring which express human antibodies. The transgenic miceare immunized using conventional methodologies with a selected antigen,e.g., all or a portion of a polypeptide of the invention. Monoclonalantibodies directed against the antigen can be obtained from theimmunized, transgenic mice using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview of this technology for producing humanantibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93,which is incorporated herein by reference in its entirety). For adetailed discussion of this technology for producing human antibodiesand human monoclonal antibodies and protocols for producing suchantibodies, see, e.g., International Publication Nos. WO 98/24893, WO96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126,5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598,which are incorporated by reference herein in their entirety. Inaddition, companies such as Abgenix, Inc. (Freemont, Calif.) and Medarex(Princeton, N.J.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules such asantibodies having a variable region derived from a non-human antibodyand a human immunoglobulin constant region. Methods for producingchimeric antibodies are known in the art. See e.g., Morrison, 1985,Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al.,1989, J. Immunol. Methods 125:191-202; and U.S. Pat. Nos. 6,311,415,5,807,715, 4,816,567, and 4,816,397, which are incorporated herein byreference in their entirety. Chimeric antibodies comprising one or moreCDRs from a non-human species and framework regions from a humanimmunoglobulin molecule can be produced using a variety of techniquesknown in the art including, for example, CDR-grafting (EP 239,400;International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539,5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106; EP519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnickaet al., 1994, Protein Engineering 7:805; and Roguska et al., 1994, Proc.Natl. Acad. Sci. USA 91:969), and chain shuffling (U.S. Pat. No.5,565,332). Each of the above-identified references is incorporatedherein by reference in its entirety.

Further, the antibodies of the invention can, in turn, be utilized togenerate anti-idiotype antibodies using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, 1989, FASEB J.7:437-444; and Nissinoff, 1991, J. Immunol. 147:2429-2438; incorporatedherein by reference in its entirety). The invention provides methodsemploying the use of polynucleotides comprising a nucleotide sequenceencoding an antibody of the invention or a fragment thereof.

The present invention encompasses single domain antibodies, includingcamelized single domain antibodies (See e.g., Muyldermans et al., 2001,Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech.1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231:25;International Publication Nos. WO 94/04678 and WO 94/25591; U.S. Pat.No. 6,005,079; which are incorporated herein by reference in theirentireties). In one embodiment, the present invention provides singledomain antibodies comprising two VH domains with modifications such thatsingle domain antibodies are formed.

The methods of the present invention also encompass the use of humanizedantibodies or fragments thereof that have half-lives (e.g., serumhalf-lives) in a mammal, preferably a human, of greater than 15 days,preferably greater than 20 days, greater than 25 days, greater than 30days, greater than 35 days, greater than 40 days, greater than 45 days,greater than 2 months, greater than 3 months, greater than 4 months, orgreater than 5 months. The increased half-lives of the humanizedantibodies of the present invention or fragments thereof in a mammal,preferably a human, results in a higher serum titer of said antibodiesor antibody fragments in the mammal, and thus, reduces the frequency ofthe administration of said antibodies or antibody fragments and/orreduces the concentration of said antibodies or antibody fragments to beadministered. Antibodies or fragments thereof having increased in vivohalf-lives can be generated by techniques known to those of skill in theart. For example, antibodies or fragments thereof with increased in vivohalf-lives can be generated by modifying (e.g., substituting, deletingor adding) amino acid residues identified as involved in the interactionbetween the Fc domain and the FcRn receptor. The humanized antibodies ofthe invention may be engineered by methods described in Ward et al. toincrease biological half-lives (See U.S. Pat. No. 6,277,375 B1;incorporated herein by reference in its entirety). For example,humanized antibodies of the invention may be engineered in the Fc-hingedomain to have increased in vivo or serum half-lives.

Antibodies or fragments thereof with increased in vivo half-lives can begenerated by attaching to said antibodies or antibody fragments polymermolecules such as high molecular weight polyethyleneglycol (PEG). PEGcan be attached to said antibodies or antibody fragments with or withouta multifunctional linker either through site-specific conjugation of thePEG to the N- or C-terminus of said antibodies or antibody fragments orvia epsilon-amino groups present on lysine residues. Linear or branchedpolymer derivatization that results in minimal loss of biologicalactivity will be used. The degree of conjugation will be closelymonitored by SDS-PAGE and mass spectrometry to ensure proper conjugationof PEG molecules to the antibodies. Unreacted PEG can be separated fromantibody-PEG conjugates by, e.g., size exclusion or ion-exchangechromatography.

The humanized antibodies of the invention may also be modified by themethods and coupling agents described by Davis et al. (See U.S. Pat. No.4,179,337; incorporated herein by reference in its entirety) in order toprovide compositions that can be injected into the mammalian circulatorysystem with substantially no immunogenic response.

The present invention also encompasses the use of humanized antibodiesor antibody fragments comprising the amino acid sequence of any of theantibodies of the invention with mutations (e.g., one or more amino acidsubstitutions) in the framework or CDR regions. Preferably, mutations inthese humanized antibodies maintain or enhance the avidity and/oraffinity of the antibodies for CD32B to which they immunospecificallybind. Standard techniques known to those skilled in the art (e.g.,immunoassays) can be used to assay the affinity of an antibody for aparticular antigen.

The invention encompasses modification of framework residues of thehumanized antibodies of the invention. Framework residues in theframework regions may be substituted with the corresponding residue fromthe CDR donor antibody to alter, preferably improve, antigen binding.These framework substitutions are identified by methods well known inthe art, e.g., by modeling of the interactions of the CDR and frameworkresidues to identify framework residues important for antigen bindingand sequence comparison to identify unusual framework residues atparticular positions. (See, e.g., U.S. Pat. No. 5,585,089; and Riechmannet al., 1988, Nature 332:323, which are incorporated herein by referencein their entireties). In certain embodiments, the invention encompassesa humanized antibody having phenylalanine at amino acid number 21 of thelight chain variable domain framework region 1 (e.g., corresponding toamino acid number 21 of SEQ ID NO:62). In other embodiments, theinvention encompasses a humanized antibody having one or more of anisoleucine at amino acid number 13 of the heavy chain variable domainframework region 2 (e.g., corresponding to amino acid number 48 of SEQID NO:60), a valine at amino acid number 6 of the heavy chain variabledomain framework region 3 (e.g., corresponding to amino acid number 72of SEQ ID NO:60), a valine at amino acid number 7 of the heavy chainvariable domain framework region 3 (e.g., corresponding to amino acidnumber 73 of SEQ ID NO:60), a valine at amino acid number 8 of the heavychain variable domain framework region 3 (e.g., corresponding to aminoacid number 74 of SEQ ID NO:60), or any combination thereof. In stillother embodiments, the invention encompasses a humanized antibody havingone or more of an isoleucine at amino acid number 13 of the heavy chainvariable domain framework region 2 (e.g., corresponding to amino acidnumber 48 of SEQ ID NO:68), a valine at amino acid number 6 of the heavychain variable domain framework region 3 (e.g., corresponding to aminoacid number 72 of SEQ ID NO:68), an aspartic acid at amino acid number 7of the heavy chain variable domain framework region 3 (e.g.,corresponding to amino acid number 73 of SEQ ID NO:68), a threonine atamino acid number 8 of the heavy chain variable domain framework region3 (e.g., corresponding to amino acid number 74 of SEQ ID NO:68), or anycombination thereof. In certain embodiments, the invention encompasseshumanized antibodies comprising at least one amino acid modification(e.g., insertion, deletion, substitution) in one or more of the lightchain variable domain framework regions. In a specific example inaccordance with this embodiment, the invention encompasses a humanizedantibody comprising a modification at amino acid 21 of the light chainvariable domain framework region 1, which modification is preferably asubstitution with phenylalanine (e.g., corresponding to amino acidnumber 21 in SEQ ID NO:62). In other embodiments, the inventionencompasses humanized antibodies comprising at least one amino acidmodification (e.g., insertion, deletion, substitution) in one or more ofthe heavy chain variable domain framework regions. In a specific examplein accordance with this embodiment, the invention encompasses ahumanized antibody comprising a modification at amino acid 13 of theheavy chain variable domain framework region 2, which modification is asubstitution with isoleucine (e.g., corresponding to amino acid number48 in SEQ ID NO:60), and/or a modification at amino acid 6 of the heavychain variable domain framework region 3, which modification is asubstitution with valine (e.g., corresponding to amino acid number 72 inSEQ ID NO:60), and/or a modification at amino acid 7 of the heavy chainvariable domain framework region 3, which modification is a substitutionwith valine (e.g., corresponding to amino acid number 73 in SEQ IDNO:60), and/or a modification at amino acid 8 of the heavy chainvariable domain framework region 3, which modification is a substitutionwith valine (e.g., corresponding to amino acid number 74 in SEQ IDNO:60). In another specific example in accordance with this embodiment,the invention encompasses a humanized antibody comprising a modificationat amino acid 13 of the heavy chain variable domain framework region 2,which modification is a substitution with isoleucine (e.g.,corresponding to amino acid number 48 in SEQ ID NO:68), and/or amodification at amino acid 6 of the heavy chain variable domainframework region 3, which modification is a substitution with valine(e.g., corresponding to amino acid number 72 in SEQ ID NO:68), and/or amodification at amino acid 7 of the heavy chain variable domainframework region 3, which modification is a substitution with asparticacid (e.g., corresponding to amino acid number 73 in SEQ ID NO:68),and/or a modification at amino acid 8 of the heavy chain variable domainframework region 3, which modification is a substitution with threonine(e.g., corresponding to amino acid number 74 in SEQ ID NO:68). Theinvention further encompasses any combination of the foregoing aminoacid modifications the heavy and or light chain variable domainframework regions.

The present invention encompasses humanized antibodies comprisingmodifications preferably, in the Fc region that modify the bindingaffinity of the antibody to one or more FcγR. Methods for modifyingantibodies with modified binding to one or more FcγR are known in theart, see, e.g., International Publication Nos. WO 04/063351, WO04/029207, WO 04/029092, WO 04/028564, WO 99/58572, WO 99/51642, WO98/23289, WO 89/07142, WO 88/07089; U.S. Publication serial Nos.2005/0037000; and 2005/0064514 and U.S. Pat. Nos. 5,843,597 and5,642,821, each of which is incorporated herein by reference in theirentirety. The invention encompasses any of the mutations disclosed inInternational Publication Nos. WO 04/063351, WO 04/029207, WO 04/029092,WO 04/028564, WO 99/58572, WO 99/51642, WO 98/23289, WO 89/07142, WO88/07089; U.S. Patent Application Publication Nos. 2005/0037000; and2005/0064514 and U.S. Pat. Nos. 5,843,597 and 5,642,821, each of whichis incorporated herein by reference in their entirety. The inventionalso encompasses any of the mutations disclosed in U.S. patentapplication Ser. Nos. 10/902,588 (filed Jul. 28, 2004) and 11/271,140(filed Nov. 10, 2005), and U.S. Provisional Application Nos. 60/707,419;and 60/781,564 filed on Aug. 10, 2005, and Mar. 10, 2006, respectively,each of which is incorporated herein by reference in their entirety. Insome embodiments, the invention encompasses antibodies that have alteredaffinity for an activating FcγR, e.g., FcγRIIIA Preferably suchmodifications also have an altered Fc-mediated effector function.Modifications that affect Fc-mediated effector function are well knownin the art (See U.S. Pat. No. 6,194,551, which is incorporated herein byreference in its entirety). In a specific embodiment, the amino acidsthat can be modified in accordance with the method of the inventioninclude, but are not limited to, Proline 329, Proline 331, and Lysine322. In certain embodiments, Proline 329, Proline 331 and Lysine 322 arereplaced with alanine; however, substitution with any other amino acidis contemplated. See International Publication No.: WO 00/42072 and U.S.Pat. No. 6,194,551 which are incorporated herein by reference in theirentirety. In preferred embodiments, the amino acids that are modified inaccordance with the methods of the invention comprise the amino acids atpositions 243, 292, 300, 305 and 396; in a specific example inaccordance with this embodiment, the amino acids that are modified arephenylalanine 243, arginine 292, tyrosine 300, valine 305 and proline396, and are preferably replaced with leucine, proline, leucine,isoleucine and leucine, respectively.

In one particular embodiment, the modification of the Fc regioncomprises one or more mutations in the Fc region. The one or moremutations in the Fc region may result in an antibody with an alteredantibody-mediated effector function, an altered binding to other Fcreceptors (e.g., Fc activation receptors), an altered ADCC activity, analtered Clq binding activity, an altered complement dependentcytotoxicity activity, an altered phagocytic activity, or anycombination thereof.

The invention also provides humanized antibodies with alteredoligosaccharide content. Oligosaccharides, as used herein, refer tocarbohydrates containing two or more simple sugars and the two terms maybe used interchangeably herein. Carbohydrate moieties of the instantinvention will be described with reference to commonly used nomenclaturein the art. For a review of carbohydrate chemistry, see, e.g., Hubbardet al., 1981 Ann. Rev. Biochem., 50: 555-583, which is incorporatedherein by reference in its entirety. This nomenclature includes, forexample, Man which represents mannose; GlcNAc which represents2-N-acetylglucosamine; Gal which represents galactose; Fuc for fucoseand Glc for glucose. Sialic acids are described by the shorthandnotation NeuNAc for 5-N-acetylneuraminic acid, and NeuNGc for5-glycolneuraminic.

In general, antibodies contain carbohydrate moeities at conservedpositions in the constant region of the heavy chain, and up to 30% ofhuman IgGs have a glycosylated Fab region. IgG has a single N-linkedbiantennary carbohydrate structure at Asn 297 which resides in the CH2domain (Jefferis et al., 1998, Immunol. Rev. 163: 59-76; Wright et al.,1997, Trends Biotech 15: 26-32). Human IgG typically has a carbohydrateof the following structure; GlcNAc(Fucose)-GlcNAc-Man-(ManGlcNAc)₂.However variations among IgGs in carbohydrate content does occur whichleads to altered function, see, e.g., Jassal et al., 2001 Biochem.Biophys. Res. Commun. 288: 243-9; Groenink et al., 1996 J. Immunol. 26:1404-7; Boyd et al., 1995 Mol. Immunol. 32: 1311-8; Kumpel et al., 1994,Human Antibody Hybridomas, 5: 143-51. The invention encompasseshumanized antibodies comprising a variation in the carbohydrate moietythat is attached to Asn 297. In one embodiment, the carbohydrate moietyhas a galactose and/or galactose-sialic acid at one or both of theterminal GlcNAc and/or a third GlcNac arm (bisecting GlcNAc). Each ofthe foregoing references is incorporated herein by reference in itsentirety.

In some embodiments, the humanized antibodies of the invention aresubstantially free of one or more selected sugar groups, e.g., one ormore sialic acid residues, one or more galactose residues, one or morefucose residues. An antibody that is substantially free of one or moreselected sugar groups may be prepared using common methods known to oneskilled in the art, including, for example, recombinantly producing anantibody of the invention in a host cell that is defective in theaddition of the selected sugar groups(s) to the carbohydrate moiety ofthe antibody, such that about 90-100% of the antibody in the compositionlacks the selected sugar group(s) attached to the carbohydrate moiety.Alternative methods for preparing such antibodies include, for example,culturing cells under conditions which prevent or reduce the addition ofone or more selected sugar groups, or post-translational removal of oneor more selected sugar groups.

In a specific embodiment, the invention encompasses a method ofproducing a substantially homogenous antibody preparation, wherein about80-100% of the antibody in the composition lacks a fucose on itscarbohydrate moiety, e.g., the carbohydrate attachment on Asn 297. Theantibody may be prepared, for example, by (a) use of an engineered hostcell that is deficient in fucose metabolism such that it has a reducedability to fucosylate proteins expressed therein; (b) culturing cellsunder conditions which prevent or reduce fusocylation; (c)post-translational removal of fucose, e.g., with a fucosidase enzyme; or(d) purification of the antibody so as to select for the product whichis not fucosylated. Most preferably, a nucleic acid encoding the desiredantibody is expressed in a host cell that has a reduced ability tofucosylate the antibody expressed therein. Preferably, the host cell isa dihydrofolate reductase deficient chinese hamster ovary cell (CHO),e.g., a Lec 13 CHO cell (lectin resistant CHO mutant cell line; (see,e.g., U.S. Patent Application Publication No. 2003/0115614; PCTPublication No. WO 00/61739; European Patent Application EP 1 229 125;Ribka & Stanley, 1986, Somatic Cell & Molec. Gen. 12(1): 51-62; Ripka etal., 1986 Arch. Biochem. Biophys. 249(2): 533-45); each of which isincorporated by reference herein in its entirety), or a CHO-K1 cell, aDUX-B11 cell, a CHO-DP12 cell or a CHO-DG44 cell, which has beenmodified so that the antibody is not substantially fucosylated. Thus,the cell may display altered expression and/or activity for thefucoysltransferase enzyme, or another enzyme or substrate involved inadding fucose to the N-linked oligosaccharide so that the enzyme has adiminished activity and/or reduced expression level in the cell. Formethods to produce antibodies with altered fucose content, see, e.g., WO03/035835 and Shields et al., 2002, J. Biol. Chem. 277(30): 26733-40;both of which are incorporated herein by reference in their entirety.

In some embodiments, the altered carbohydrate modifications modulate oneor more of the following: solubilization of the antibody, facilitationof subcellular transport and secretion of the antibody, promotion ofantibody assembly, conformational integrity, and antibody-mediatedeffector function. In a specific embodiment the altered carbohydratemodifications enhance antibody mediated effector function relative tothe antibody lacking the carbohydrate modification. Carbohydratemodifications that lead to altered antibody mediated effector functionare well known in the art (for example, see Shields R. L. et al., 2001,J. Biol. Chem. 277(30): 26733-40; Davies J. et al., 2001, Biotechnology& Bioengineering, 74(4): 288-294; each of which is incorporated hereinby reference in its entirety). In another specific embodiment, thealtered carbohydrate modifications enhance the binding of antibodies ofthe invention to FcγRIIB receptor. Altering carbohydrate modificationsin accordance with the methods of the invention includes, for example,increasing the carbohydrate content of the antibody or decreasing thecarbohydrate content of the antibody. Methods of altering carbohydratecontents are known to those skilled in the art, see, e.g., Wallick etal., 1988, Journal of Exp. Med. 168(3): 1099-1109; Tao et al., 1989Journal of Immunology, 143(8): 2595-2601; Routledge et al., 1995Transplantation, 60(8): 847-53; Elliott et al. 2003; NatureBiotechnology, 21: 414-21; Shields et al. 2002 Journal of BiologicalChemistry, 277(30): 26733-40; all of which are incorporated herein byreference in their entirety.

In some embodiments, the invention encompasses humanized antibodiescomprising one or more glycosylation sites, so that one or morecarbohydrate moieties are covalently attached to the antibody. In otherembodiments, the invention encompasses humanized antibodies comprisingone or more glycosylation sites and one or more modifications in the Fcregion, such as those disclosed supra and those known to one skilled inthe art. In preferred embodiments, the one or more modifications in theFc region enhance the affinity of the antibody for an activating FcγR,e.g., FcγRIIIA, relative to the antibody comprising the wild type Fcregions. Humanized antibodies of the invention with one or moreglycosylation sites and/or one or more modifications in the Fc regionhave an enhanced antibody mediated effector function, e.g., enhancedADCC activity. In some embodiments, the invention further compriseshumanized antibodies comprising one or more modifications of amino acidsthat are directly or indirectly known to interact with a carbohydratemoiety of the antibody, including, but not limited to, amino acids atpositions 241, 243, 244, 245, 245, 249, 256, 258, 260, 262, 264, 265,296, 299, and 301. Amino acids that directly or indirectly interact witha carbohydrate moiety of an antibody are known in the art, see, e.g.,Jefferis et al., 1995 Immunology Letters, 44: 111-7, which isincorporated herein by reference in its entirety.

The invention encompasses humanized antibodies that have been modifiedby introducing one or more glycosylation sites into one or more sites ofthe antibodies, preferably without altering the functionality of theantibody, e.g., binding activity to FcγRIIB. Glycosylation sites may beintroduced into the variable and/or constant region of the antibodies ofthe invention. As used herein, “glycosylation sites” include anyspecific amino acid sequence in an antibody to which an oligosaccharide(i.e., carbohydrates containing two or more simple sugars linkedtogether) will specifically and covalently attach. Oligosaccharide sidechains are typically linked to the backbone of an antibody via either N-or O-linkages. N-linked glycosylation refers to the attachment of anoligosaccharide moiety to the side chain of an asparagine residue.O-linked glycosylation refers to the attachment of an oligosaccharidemoiety to a hydroxyamino acid, e.g., serine, threonine. The antibodiesof the invention may comprise one or more glycosylation sites, includingN-linked and O-linked glycosylation sites. Any glycosylation site forN-linked or O-linked glycosylation known in the art may be used inaccordance with the instant invention. An exemplary N-linkedglycosylation site that is useful in accordance with the methods of thepresent invention, is the amino acid sequence: Asn-X-Thr/Ser, wherein Xmay be any amino acid and Thr/Ser indicates a threonine or a serine.Such a site or sites may be introduced into an antibody of the inventionusing methods well known in the art to which this invention pertains.See, for example, “In Vitro Mutagenesis,” Recombinant DNA: A ShortCourse, J. D. Watson, et al. W.H. Freeman and Company, New York, 1983,chapter 8, pp. 106-116, which is incorporated herein by reference in itsentirety. An exemplary method for introducing a glycosylation site intoan antibody of the invention may comprise: modifying or mutating anamino acid sequence of the antibody so that the desired Asn-X-Thr/Sersequence is obtained.

In some specific embodiments, the invention encompasses modifiedhumanized FcγRIIB antibodies wherein the N-glysosylation consensus siteAsn₅₀-Val-Ser of the CDR2 region has been modified, so that theglycosylation site at position 50 is eliminated. Although not intendingto be bound by a particular mechanism of action, removal of theglycosylation site may limit potential variation in production of theantibody as well as potential immunogenicity in a pharmaceuticalapplication. In a specific embodiment, the invention encompasses ahumanized FcγRIIB antibody wherein the amino acid at position 50 hasbeen modified, e.g., deleted or substituted. In another specificembodiment, the invention further encompasses an amino acidmodification, e.g., deletion or substitution, at position 51. In onespecific embodiment, the invention encompasses a humanized FcγRIIBantibody wherein the amino acid at position 50 has been replaced withtyrosine. In another more specific embodiment, the invention encompassesa humanized FcγRIIB antibody wherein the amino acid at position 50 hasbeen replaced with tyrosine and the amino acid at position 51 has beenreplaced with alanine.

In some embodiments, the invention encompasses methods of modifying thecarbohydrate content of an antibody of the invention by adding ordeleting a glycosylation site. Methods for modifying the carbohydratecontent of antibodies are well known in the art and encompassed withinthe invention, see, e.g., U.S. Pat. No. 6,218,149; EP 0 359 096 B1; U.S.Patent Application Publication No. US 2002/0028486; WO 03/035835; U.S.Publication No. 2003/0115614; U.S. Pat. No. 6,218,149; U.S. Pat. No.6,472,511; all of which are incorporated herein by reference in theirentirety. In other embodiments, the invention encompasses methods ofmodifying the carbohydrate content of an antibody of the invention bydeleting one or more endogenous carbohydrate moieties of the antibody.

The invention further encompasses methods of modifying an effectorfunction of an antibody of the invention, wherein the method comprisesmodifying the carbohydrate content of the antibody using the methodsdisclosed herein or known in the art.

Standard techniques known to those skilled in the art can be used tointroduce mutations in the nucleotide sequence encoding an antibody, orfragment thereof, including, e.g., site-directed mutagenesis andPCR-mediated mutagenesis, which results in amino acid substitutions.Preferably, the derivatives include less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the original antibody or fragment thereof. In a preferredembodiment, the derivatives have conservative amino acid substitutionsmade at one or more predicted non-essential amino acid residues.

The present invention also encompasses humanized antibodies or fragmentsthereof comprising an amino acid sequence of a variable heavy chainand/or variable light chain that is at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 99% identicalto the amino acid sequence of the variable heavy chain and/or lightchain of the mouse monoclonal antibody produced by clone 2B6, 3H7, 1D5,2E1, 2H9, 2D11, or 1F2, with ATCC accession numbers PTA-4591, PTA-4592,PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively. Inpreferred embodiments of the invention, the humanized antibody orfragemnt thereof comprises a heavy chain having the amino acid sequenceSEQ ID NO:70 and/or light chain having the amino acid sequence SEQ IDNO:66. The present invention further encompasses antibodies or fragmentsthereof that specifically bind FcγRIIB with greater affinity than saidantibody or fragment thereof binds FcγRIIA and/or bind to FcγRIIB andblock the Fc binding domain of FcγRIIB, said antibodies or antibodyfragments comprising an amino acid sequence of one or more CDRs that isat least 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99% identical to the amino acid sequence of oneor more CDRs of the mouse monoclonal antibody produced by clone 2B6,3H7, 1D5, 2E1, 2H9, 2D11, or 1F2, with ATCC accession numbers PTA-4591,PTA-4592, PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959,respectively. The determination of percent identity of two amino acidsequences can be determined by any method known to one skilled in theart, including BLAST protein searches.

The present invention also encompasses the use of humanized antibodiesor antibody fragments that specifically bind FcγRIIB with greateraffinity than said antibodies or fragments thereof binds FcγRIIA and/orbind to FcγRIIB and block the Fc binding domain of FcγRIIB, wherein saidantibodies or antibody fragments are encoded by a nucleotide sequencethat hybridizes to the nucleotide sequence of the mouse monoclonalantibody produced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2, withATCC accession numbers PTA-4591, PTA-4592, PTA-5958, PTA-5961, PTA-5962,PTA-5960, and PTA-5959, respectively, under stringent conditions. In aspecific embodiments, the invention encompasses the use of humanizedantibodies or antibody fragments that specifically bind FcγRIIB withgreater affinity than said antibodies or fragments thereof binds FcγRIIAand/or bind to FcγRIIB and block the Fc binding domain of FcγRIIB,wherein said antibodies or antibody fragments are encoded by anucleotide sequence that hybridizes under stringent conditions to thenucleotide sequence encoding a heavy chain variable domain or thecomplete heavy chain of a humainized 2B6 antibody of the invention,e.g., SEQ ID NO:67 or SEQ ID NO:69, respectively. In yet otherembodiments, the invention encompasses the use of humanized antibodiesor antibody fragments that specifically bind FcγRIIB with greateraffinity than said antibodies or fragments thereof binds FcγRIIA and/orbind to FcγRIIB and block the Fc binding domain of FcγRIIB, wherein saidantibodies or antibody fragments are encoded a nucleotide sequence thathybridizes under stringent conditions to the nucleotide sequenceencoding the light chain variable domain or the light chain of ahumanized 2B6 antibody of the invention, e.g., SEQ ID NO:61 or SEQ IDNO:65, respectively. In a preferred embodiment, the invention providesantibodies or fragments thereof that specifically bind FcγRIIB withgreater affinity than said antibodies or fragments thereof bind FcγRIIA,said antibodies or antibody fragments comprising a variable light chainand/or variable heavy chain encoded by a nucleotide sequence thathybridizes under stringent conditions to the nucleotide sequence of thevariable light chain and/or variable heavy chain of the mouse monoclonalantibody produced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2, withATCC accession numbers PTA-4591, PTA-4592, PTA-5958, PTA-5961, PTA-5962,PTA-5960, and PTA-5959, respectively, under stringent conditions. Inanother preferred embodiment, the invention provides antibodies orfragments thereof that specifically bind FcγRIIB with greater affinitythan said antibodies or fragments thereof bind FcγRIIA, said antibodiesor antibody fragments comprising one or more CDRs encoded by anucleotide sequence that hybridizes under stringent conditions to thenucleotide sequence of one or more CDRs of the mouse monoclonal antibodyproduced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2, with ATCCaccession numbers PTA-4591, PTA-4592, PTA-5958, PTA-5961, PTA-5962,PTA-5960, and PTA-5959, respectively. Stringent hybridization conditionsinclude, but are not limited to, hybridization to filter-bound DNA in 6×sodium chloride/sodium citrate (SSC) at about 45° C. followed by one ormore washes in 0.2×SSC/0.1% SDS at about 50-65° C., highly stringentconditions such as hybridization to filter-bound DNA in 6×SSC at about45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 60°C., or any other stringent hybridization conditions known to thoseskilled in the art (see, for example, Ausubel, F. M. et al., eds. 1989Current Protocols in Molecular Biology, vol. 1, Green PublishingAssociates, Inc. and John Wiley and Sons, Inc., NY at pages 6.3.1 to6.3.6 and 2.10.3, incorporated herein by reference).

6.1.1 Antibody Conjugates

The present invention encompasses humanized antibodies recombinantlyfused or chemically conjugated (including both covalently andnon-covalently conjugations) to heterologous polypeptides (i.e., anunrelated polypeptide; or portion thereof, preferably at least 10, atleast 20, at least 30, at least 40, at least 50, at least 60, at least70, at least 80, at least 90 or at least 100 amino acids of thepolypeptide) to generate fusion proteins. The fusion does notnecessarily need to be direct, but may occur through linker sequences.Humanized antibodies may be used for example to target heterologouspolypeptides to particular cell types, either in vitro or in vivo, byfusing or conjugating the antibodies to antibodies specific forparticular cell surface receptors. Antibodies fused or conjugated toheterologous polypeptides may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., PCTPublication No. WO 93/21232; EP 439,095; Naramura et al., 1994 Immunol.Lett., 39:91-99; U.S. Pat. No. 5,474,981; Gillies et al., 1992 Proc.Natl. Acad. Sci. USA, 89:1428-1432; and Fell et al., 1991, J. Immunol.,146:2446-2452, all of which are incorporated herein by reference intheir entireties.

Further, a humanized antibody may be conjugated to a therapeutic agentor drug moiety that modifies a given biological response. Therapeuticagents or drug moieties are not to be construed as limited to classicalchemical therapeutic agents. For example, the drug moiety may be aprotein or polypeptide possessing a desired biological activity. Suchproteins may include, for example, a toxin such as abrin, ricin A,pseudomonas exotoxin (i.e., PE-40), or diphtheria toxin, ricin, gelonin,and pokeweed antiviral protein, a protein such as tumor necrosis factor,interferons including, but not limited to, α-interferon (IFN-α),β-interferon (IFN-β), nerve growth factor (NGF), platelet derived growthfactor (PDGF), tissue plasminogen activator (TPA), an apoptotic agent(e.g., TNF-α, TNF-β, AIM I as disclosed in PCT Publication No. WO97/33899), AIM II (see, e.g., PCT Publication No. WO 97/34911), FasLigand (Takahashi et al., 1994 J. Immunol., 6:1567-1574), and VEGI (PCTPublication No. WO 99/23105), a thrombotic agent or an anti-angiogenicagent (e.g., angiostatin or endostatin), or a biological responsemodifier such as, for example, a lymphokine (e.g., interleukin-1(“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocytemacrophage colony stimulating factor (“GM-CSF”), and granulocyte colonystimulating factor (“G-CSF”)), macrophage colony stimulating factor,(“M-CSF”), or a growth factor (e.g., growth hormone (“GH”); a protease,or a ribonuclease. Each of the foregoing references is incorporatedherein by reference in its entirety.

Humanized antibodies can be fused to marker sequences, such as apeptide, to facilitate purification. In preferred embodiments, themarker amino acid sequence is a hexa-histidine peptide, such as the tagprovided in a pQE™ vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,Calif., 91311), among others, many of which are commercially available.As described in Gentz et al., 1989 Proc. Natl. Acad. Sci. USA,86:821-824 (incorporated herein by reference in its entirety), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Other peptide tags useful for purification include, butare not limited to, the hemagglutinin “HA” tag, which corresponds to anepitope derived from the influenza hemagglutinin protein (Wilson et al.,1984 Cell, 37:767) and the “flag” tag (Knappik et al., 1994Biotechniques, 17(4):754-761) (both of which references are incorporatedherein by reference in its entirety).

The present invention further includes compositions comprisingheterologous polypeptides fused or conjugated to antibody fragments. Forexample, the heterologous polypeptides may be fused or conjugated to aFab fragment, Fd fragment, Fv fragment, F(ab)_(z) fragment, or portionthereof. Methods for fusing or conjugating polypeptides to antibodyportions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603,5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; EP 307,434;EP 367,166; International Publication Nos. WO 96/04388 and WO 91/06570;Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539;Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vil et al., 1992,Proc. Natl. Acad. Sci. USA 89:11337-11341 (said references incorporatedby reference in their entireties).

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of antibodies of the invention orfragments thereof (e.g., antibodies or fragments thereof with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten etal., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, TrendsBiotechnol. 16:76; Hansson, et al., 1999, J. Mol. Biol. 287:265; andLorenzo and Blasco, 1998, BioTechniques 24:308 (each of these patentsand publications are hereby incorporated by reference in its entirety).Antibodies or fragments thereof, or the encoded antibodies or fragmentsthereof, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. One or more portions of a polynucleotide encoding anantibody or antibody fragment, which portions specifically bind toFcγRIIB may be recombined with one or more components, motifs, sections,parts, domains, fragments, etc. of one or more heterologous molecules.

The present invention also encompasses humanized antibodies conjugatedto a diagnostic or therapeutic agent or any other molecule for whichserum half-life is desired to be increased. The humanized antibodies canbe used diagnostically to, for example, monitor the development orprogression of a disease, disorder or infection as part of a clinicaltesting procedure to, e.g., determine the efficacy of a given treatmentregimen. Detection can be facilitated by coupling the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody or indirectly, through an intermediate (such as, for example, alinker known in the art) using techniques known in the art. See, forexample, U.S. Pat. No. 4,741,900 (incorporated herein by reference inits entirety) for metal ions which can be conjugated to antibodies foruse as diagnostics according to the present invention. Such diagnosisand detection can be accomplished by coupling the antibody to detectablesubstances including, but not limited to, various enzymes, enzymesincluding, but not limited to, horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase; prostheticgroup complexes such as, but not limited to, streptavidin/biotin andavidin/biotin; fluorescent materials such as, but not limited to,umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent material such as, but not limited to, luminol;bioluminescent materials such as, but not limited to, luciferase,luciferin, and aequorin; radioactive material such as, but not limitedto, bismuth (²¹³Bi), carbon (¹⁴C), chromium (⁵¹Cr), cobalt (⁵⁷Co),fluorine (¹⁸F), gadolinium (¹⁵³Gd, ¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga),germanium (⁶⁸Ge), holmium (¹⁶⁶Ho), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹I),iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), lanthanium, (¹⁴⁰La), lutetium (¹⁷⁷Lu),manganese (⁵⁴Mn), molybdenum (⁹⁹Mo), palladium (¹⁰³Pd), phosphorous(³²P), praseodymium (¹⁴²Pr), promethium (¹⁴⁹Pm), rhenium ¹⁸⁶Re, ¹⁸⁸Re),rhodium (¹⁰⁵Rh), ruthemium (⁹⁷Ru), samarium (¹⁵³Sm), scandium (⁴⁷Sc),selenium (⁷⁵Se), strontium (⁸⁵Sr), sulfur (³⁵S), technetium (⁹⁹Tc),thallium (²⁰¹Ti), tin (¹¹³Sn, ¹¹⁷Sn), tritium (³H), xenon (¹³³Xe),ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb), yttrium (⁹⁰Y), zinc (⁶⁵Zn); positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions.

An antibody may be conjugated to a therapeutic moiety such as acytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agentor a radioactive element (e.g., alpha-emitters, gamma-emitters, etc.).Cytotoxins or cytotoxic agents include any agent that is detrimental tocells. Examples include paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC)), and anti-mitotic agents (e.g., vincristine andvinblastine).

Moreover, a humanized antibody can be conjugated to therapeutic moietiessuch as a radioactive materials or macrocyclic chelators useful forconjugating radiometal ions (see above for examples of radioactivematerials). In certain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4:2483-90; Peterson et al., 1999, Bioconjug.Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943-50each incorporated by reference in their entireties.

Techniques for conjugating such therapeutic moieties to antibodies arewell known; see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), 1985, pp. 243-56, Alan R.Liss, Inc.); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), 1987, pp.623-53, Marcel Dekker, Inc.); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), 1985, pp.475-506); “Analysis, Results, And Future Prospective Of The TherapeuticUse Of Radiolabeled Antibody In Cancer Therapy”, in MonoclonalAntibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),1985, pp. 303-16, Academic Press; and Thorpe et al., Immunol. Rev.,62:119-58, 1982; each incorporated by reference in its entirety.

An antibody or fragment thereof, with or without a therapeutic moietyconjugated to it, administered alone or in combination with cytotoxicfactor(s) and/or cytokine(s) can be used as a therapeutic.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

6.2 Preparation of FcγRIIB Humanized Antibodies

The invention encompasses nucleotide sequences that encode theCDR-grafted heavy and light chains, cloning and expression vectorscontaining the nucleotide sequences, host cells transformed with thenucleotide sequences, and methods for the production of the CDR-graftedchains and antibody molecules comprising the nucleotide sequences in thetransformed host cells. In specific embodiments, the inventionencompasses any of the nucleotide sequences of SEQ ID NOS. 17, 19, 21,23, 36 or 45.

The invention encompasses donor amino acid sequences, which encodeantibodies that bind FcγRIIB with a greater affinity that FcγRIIA, suchas those disclosed in U.S. Provisional Application No. 60/403,366, filedon Aug. 14, 2002 and U.S. Patent Application Publication No.2004/0185045, both of which are incorporated herein by reference intheir entireties. In a specific embodiment, the donor amino acidsequence encodes for the monoclonal antibody produced from clone 2B6,3H7, 1D5, 2E1, 2H9, 2D11, or 1F2, with ATCC accession numbers PTA-4591,PTA-4592, PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959,respectively, or other monoclonal antibodies produced by immunizationmethods of the invention as disclosed in U.S. Provisional ApplicationNo. 60/403,366, filed on Aug. 14, 2002 and U.S. Patent ApplicationPublication No. 2004/0185045, both of which are incorporated herein byreference in their entireties. The invention also encompasspolynucleotides that encode for donor amino acid sequences thathybridize under various stringency, e.g., high stringency, intermediateor low stringency conditions, to polynucleotides that encode for themonoclonal antibody produced from clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11,or 1F2, with ATCC accession numbers PTA-4591, PTA-4592, PTA-5958,PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively, or othermonoclonal antibodies produced by immunization methods of the inventionas disclosed in U.S. Provisional Application No. 60/403,366, filed onAug. 14, 2002 and U.S. Patent Application Publication No. 2004/0185045;both of which are incorporated herein by reference in their entirety.The hybridization can be performed under various conditions ofstringency. By way of example and not limitation, procedures usingconditions of low stringency are as follows (see also Shilo andWeinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 6789-6792;incorporated herein by reference in its entirety). Filters containingDNA are pretreated for 6 h at 40° C. in a solution containing 35%formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1%Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizationsare carried out in the same solution with the following modifications:0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10%(wt/vol) dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe is used.Filters are incubated in hybridization mixture for 18-20 h at 40° C.,and then washed for 1.5 h at 55° C. in a solution containing 2×SSC, 25mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 h at 60° C.Filters are blotted dry and exposed for autoradiography. If necessary,filters are washed for a third time at 65-68° C. and re-exposed to film.Other conditions of low stringency which may be used are well known inthe art (e.g., as employed for cross-species hybridizations). By way ofexample and not limitation, procedures using conditions of highstringency are as follows. Prehybridization of filters containing DNA iscarried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC,50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA,and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48h at 65° C. in prehybridization mixture containing 100 μg/ml denaturedsalmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Washing offilters is done at 37° C. for 1 h in a solution containing 2×SSC, 0.01%PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSCat 50° C. for 45 min before autoradiography. Other conditions of highstringency which may be used are well known in the art. Selection ofappropriate conditions for such stringencies is well known in the art(see e.g., Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; see also, Ausubel et al., eds., in the Current Protocols inMolecular Biology series of laboratory technique manuals, ©1987-1997,Current Protocols, ©1994-1997 John Wiley and Sons, Inc.; see especially,Dyson, 1991, “Immobilization of nucleic acids and hybridizationanalysis,” In: Essential Molecular Biology: A Practical Approach, Vol.2, T. A. Brown, ed., pp. 111-156, IRL Press at Oxford University Press,Oxford, UK; each of which reference is incorporated herein by referencein its entirety). The polynucleotides may be obtained, and thenucleotide sequence of the polynucleotides determined, by any methodknown in the art.

DNA sequences which encode the acceptor amino acid sequences may beobtained by any method known to one skilled in the art. For example, DNAsequences coding for preferred human acceptor framework sequencesinclude but are not limited to FR segments from the human germline VHsegment VH1-8 and JH6 and the human germline VL segment VK-A26 and JK4.

In a specific embodiment, one or more of the CDRs are inserted withinframework regions using routine recombinant DNA techniques. Theframework regions may be naturally occurring or consensus frameworkregions, and preferably human framework regions (see, e.g., Chothia etal., 1998, J. Mol. Biol. 278: 457-479 for a listing of human frameworkregions; incorporated herein by reference in its entirety). Preferably,the polynucleotide generated by the combination of the framework regionsand CDRs encodes an antibody that specifically binds to FcγRIIB withgreater affinity than said antibody binds FcγRIIA. Preferably, asdiscussed supra, one or more amino acid substitutions may be made withinthe framework regions, and, preferably, the amino acid substitutionsimprove binding of the antibodies of the invention to FcγRIIB.

In another embodiment, human libraries or any other libraries availablein the art, can be screened by standard techniques known in the art, toclone the nucleic acids encoding the antibodies of the invention.

The humanized antibodies of the present invention may be produced by anymethod known in the art useful for the production of polypeptides, e.g.,in vitro synthesis, recombinant DNA production, and the like.Preferably, the humanized antibodies are produced by recombinant DNAtechnology. The humanized FcγRIIB specific antibodies of the inventionmay be produced using recombinant immunoglobulin expression technology.The recombinant production of immunoglobulin molecules, includinghumanized antibodies are described in U.S. Pat. No. 4,816,397 (Boss etal.), U.S. Pat. Nos. 6,331,415 and 4,816,567 (both to Cabilly et al.),U.K. patent GB 2,188,638 (Winter et al.), and U.K. patent GB 2,209,757;all of which are incorporated herein by reference in their entireties.Techniques for the recombinant expression of immunoglobulins, includinghumanized immunoglobulins, can also be found, in Goeddel et al., GeneExpression Technology Methods in Enzymology Vol. 185 Academic Press(1991), and Borreback, Antibody Engineering, W. H. Freeman (1992) (bothof which are incorporated herein by reference in their entirety.Additional information concerning the generation, design and expressionof recombinant antibodies can be found in Mayforth, DesigningAntibodies, Academic Press, San Diego (1993).

An exemplary process for the production of the recombinant humanizedantibodies of the invention may comprise the following: a) constructing,by conventional molecular biology methods, an expression vectorcomprising an operon that encodes an antibody heavy chain in which theCDRs and a minimal portion of the variable region framework that arerequired to retain donor antibody binding specificity are derived from anon-human immunoglobulin, such as the murine FcγRIIB monoclonalantibody, and the remainder of the antibody is derived from a humanimmunoglobulin, thereby producing a vector for the expression of ahumanized antibody heavy chain; b) constructing, by conventionalmolecular biology methods, an expression vector comprising an operonthat encodes an antibody light chain in which the CDRs and a minimalportion of the variable region framework that are required to retaindonor antibody binding specificity are derived from a non-humanimmunoglobulin, such as the murine FcγRIIB monoclonal antibody, and theremainder of the antibody is derived from a human immunoglobulin,thereby producing a vector for the expression of humanized antibodylight chain; c) transferring the expression vectors to a host cell byconventional molecular biology methods to produce a transfected hostcell for the expression of humanized anti-FcγRIIB antibodies; and d)culturing the transfected cell by conventional cell culture techniquesso as to produce humanized anti-FcγRIIB antibodies. Host cells may becotransfected with two expression vectors of the invention, the firstvector containing an operon encoding a heavy chain derived polypeptideand the second containing an operon encoding a light chain derivedpolypeptide. The two vectors may contain different selectable markersbut, with the exception of the heavy and light chain coding sequences,are preferably identical. This procedure provides for equal expressionof heavy and light chain polypeptides. Alternatively, a single vectormay be used which encodes both heavy and light chain polypeptides. Thecoding sequences for the heavy and light chains may comprise cDNA orgenomic DNA or both. The host cell used to express the recombinantantibody of the invention may be either a bacterial cell such asEscherichia coli, or preferably a eukaryotic cell. Preferably, amammalian cell such as a chinese hamster ovary cell or HEK-293 cells,may be used. The choice of expression vector is dependent upon thechoice of host cell, and may be selected so as to have the desiredexpression and regulatory characteristics in the selected host cell.Other cell lines that may be used include, but are not limited to,CHO-K1, NSO, and PER.C6 (Crucell, Leiden, Netherlands).

In a specific embodiment the method for producing a humanized FcγRIIB2B6 antibody comprises the following: RNA from hybridoma cells of 2B6 isconverted to cDNA and the VH and VL segments are PCR amplified using,for example, the RLM-RACE kit (Ambion, Inc.). Gene specific primers forthe VH are used. Examples of such primers for VH include: SJ15R, SEQ IDNO: 47 (5′ GGT CAC TGT CAC TGG CTC AGG G 3′) and SJ16R, SEQ ID NO: 48(5′ AGG CGG ATC CAG GGG CCA GTG GAT AGA C3′), and for VL include SJ17R,SEQ ID NO: 49 (5′ GCA CAC GAC TGA GGC ACC TCC AGA TG 3′) and SJ18R, SEQID NO. 50 (5′ CGG CGG ATC CGA TGG ATA CAG TTG GTG CAG CAT C3′). The RACEproduct is inserted into a plasmid, e.g., pCR2.1-TOPO using a TOPO TACloning kit (Invitrogen, Inc.). The resulting plasmids are thensubjected to DNA sequencing to determine the VH and VL sequences for2B6. The resulting sequences are translated and the predicted amino acidsequence determined for each. From these sequences the framework (FR)and complementarity determining (CDR) regions are identified as definedby Kabat. The mouse VH is then joined to a human C-Gammal constantregion and an Ig leader sequence and inserted into pCI-neo for mammalianexpression. The mouse VL is joined to a human C-kappa segment and an Igleader sequence and also cloned into pCI-neo for mammalian expression.The humanized 2B6 VH consists of the FR segments from the human germlineVH segment VH1-18 and JH6, and the CDR regions of the 2B6 VH. Thehumanized 2B6 VL consists of the FR segments of the human germline VLsegment VK-A26 and JK4, and the CDR regions of 2B6 VL. The humanized VHand VL segments are assembled de novo from oligonucleotides combined andamplified by PCR. The resulting fragment is then combined by PCR with aleader sequence and the appropriate constant region segment cloned intothe expression vector pCI-neo. The DNA sequence of the resultingplasmids is confirmed by sequence analysis. After this procedure lightchain segments having predicted humanized 2B6 VL sequence areidentified. Representative plasmids, pMGx608 (containing a humanized 2B6heavy chain) and pMGx611 (containing a humanized 2B6 light chain withN₅₀→Y and V₅₁→A in CDR2), having ATCC Accession numbers PTA-5963 andPTA-5964, respectively, were deposited under the provisions of theBudapest Treaty with the American Type Culture Collection (10801University Blvd., Manassas, Va. 20110-2209) on May 7, 2004,respectively, and are incorporated herein by reference.

The general methods for construction of the vectors of the invention,transfection of cells to produce the host cell of the invention, cultureof cells to produce the antibody of the invention are all conventionalmolecular biology methods. Likewise, once produced, the recombinanthumanized antibodies of the invention may be purified by standardprocedures of the art, including cross-flow filtration, ammoniumsulphate precipitation, affinity column chromatography, gelelectrophoresis and the like.

The humanized FcγRIIB specific antibodies of the present invention maybe used in conjunction with, or attached to, other antibodies (or partsthereof) such as human or humanized monoclonal antibodies. These otherantibodies may be reactive with other markers (epitopes) characteristicfor the disease against which the antibodies of the invention aredirected or may have different specificities chosen, for example, torecruit molecules or cells of the human immune system to the diseasedcells. The antibodies of the invention (or parts thereof) may beadministered with such antibodies (or parts thereof) as separatelyadministered compositions or as a single composition with the two agentslinked by conventional chemical or by molecular biological methods.Additionally the diagnostic and therapeutic value of the antibodies ofthe invention may be augmented by labelling the humanized antibodieswith labels that produce a detectable signal (either in vitro or invivo) or with a label having a therapeutic property. Some labels, e.g.,radionucleotides, may produce a detectable signal and have a therapeuticproperty. Examples of radionuclide labels include, but are not limitedto, ¹²⁵I, ¹³¹I, and ¹⁴C. Examples of other detectable labels include afluorescent chromophore such as fluorescein, phycobiliprotein ortetraethyl rhodamine for fluorescence microscopy, an enzyme whichproduces a fluorescent or colored product for detection by fluorescence,absorbance, visible color or agglutination, which produces an electrondense product for demonstration by electron microscopy; or an electrondense molecule such as ferritin, peroxidase or gold beads for direct orindirect electron microscopic visualization. Labels having therapeuticproperties include drugs for the treatment of cancer, such asmethotrexate and the like.

The subject invention provide numerous humanized antibodies specific forthe FcγRIIB based on the discovery that the CDR regions of the murinemonoclonal antibody could be spliced into a human acceptor framework soas to produce a humanized recombinant antibody specific for the FcγRIIB.Preferred humanized FcγRIIB specific antibodies contain an additionalchange in the framework region (or in other regions) to increasingbinding for FcγRIIB. Particularly preferred embodiments of the inventionare the exemplified humanized antibody molecules that have superiorbinding properties for FcγRIIB.

The invention encompasses standard recombinant DNA methods for preparingDNA sequences which code for the CDR-grafted antibodies of theinvention. DNA sequences may be synthesized completely or in part usingoligonucleotide synthesis techniques. Methods for oliogonucleotidedirected synthesis are well known in the art. The invention furtherencompasses site-directed mutagenesis methods such as those known in theart.

Any suitable host cell/vector system may be used for expression of theDNA sequences coding for the CDR-grafted heavy and light chains.Bacterial, e.g., E. coli, and other microbial systems may be used, inparticular for expression of antibody fragments such as Fab and (Fab′)2fragments, and especially FV fragments and single chain antibodyfragments, e.g., single chain FVs. Eucaryotic systems, e.g., mammalianhost cell expression systems, may be used for production of largerCDR-grafted antibody products, including complete antibody molecules.Suitable mammalian host cells include CHO cells and myeloma or hybridomacell lines. Other cell lines that may be used include, but are notlimited to, CHO-K1, NSO, and PER.C6 (Crucell, Leiden, Netherlands).

The donor murine antibodies of the invention may be produced using anymethod known in the art, including those disclosed in U.S. PatentApplication Publication Nos. 2004/0185045; 2005/02157667; 2005/060213;and 2006/0013810; International Publication Nos. WO 04/016750; WO2005/110474; and WO 2005/115452; and Provisional Application No.60/636,663 (filed Dec. 15, 2004); each of which are incorporated hereinby reference in their entireties.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments may be producedby proteolytic cleavage of immunoglobulin molecules, using enzymes suchas papain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). F(ab′)₂ fragments contain the complete light chain, and thevariable region, the CH1 region and at least a portion of the hingeregion of the heavy chain.

For example, antibodies can also be generated using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of phage particles whichcarry the polynucleotide sequences encoding them. In a particularembodiment, such phage can be utilized to display antigen bindingdomains, such as Fab and Fv or disulfide-bond stabilized Fv, expressedfrom a repertoire or combinatorial antibody library (e.g., human ormurine). Phage expressing an antigen binding domain that binds theantigen of interest can be selected or identified with antigen, e.g.,using labeled antigen or antigen bound or captured to a solid surface orbead. Phage used in these methods are typically filamentous phage,including fd and M13. The antigen binding domains are expressed as arecombinantly fused protein to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theimmunoglobulins, or fragments thereof, of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods, 182:41-50,1995; Ames et al., J. Immunol. Methods, 184:177-186, 1995; Kettleboroughet al., Eur. J. Immunol., 24:952-958, 1994; Persic et al., Gene,187:9-18, 1997; Burton et al., Advances in Immunology, 57:191-280, 1994;PCT Application No. PCT/GB91/01134; PCT Publications WO 90/02809; WO91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which isincorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired fragments, and expressed in any desired host, includingmammalian cells, insect cells, plant cells, yeast, and bacteria, e.g.,as described in detail below. For example, techniques to recombinantlyproduce Fab, Fab′ and F(ab′)₂ fragments can also be employed usingmethods known in the art such as those disclosed in PCT Publication WO92/22324; Mullinax et al., BioTechniques, 12(6):864-869, 1992; and Sawaiet al., AJRI, 34:26-34, 1995; and Better et al., Science, 240:1041-1043,1988 (each of which is incorporated by reference in its entirety).Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology, 203:46-88, 1991; Shu etal., Proc. Natl. Acad. Sci. USA, 90:7995-7999, 1993; and Skerra et al.,Science, 240:1038-1040, 1988 (each of which is incorporated by referencein its entirety).

Phage display technology can be used to increase the affinity of anantibody of the invention for FcγRIIB. This technique would be useful inobtaining high affinity antibodies that could be used in thecombinatorial methods of the invention. This technology, referred to asaffinity maturation, employs mutagenesis or CDR walking and re-selectionusing FcγRIIB or an antigenic fragment thereof to identify antibodiesthat bind with higher affinity to the antigen when compared with theinitial or parental antibody (See, e.g., Glaser et al., 1992, J.Immunology 149:3903; incorporated by reference in its entirety).Mutagenizing entire codons rather than single nucleotides results in asemi-randomized repertoire of amino acid mutations. Libraries can beconstructed consisting of a pool of variant clones each of which differsby a single amino acid alteration in a single CDR and which containvariants representing each possible amino acid substitution for each CDRresidue. Mutants with increased binding affinity for the antigen can bescreened by contacting the immobilized mutants with labeled antigen. Anyscreening method known in the art can be used to identify mutantantibodies with increased avidity to the antigen (e.g., ELISA) (See Wuet al., 1998, Proc Natl. Acad. Sci. USA 95:6037; Yelton et al., 1995, J.Immunology 155:1994; each of which is incorporated by reference in itsentirety). CDR walking which randomizes the light chain is also possible(See Schier et al., 1996, J. Mol. Bio. 263:551; incorporated byreference in its entirety).

6.2.1 Screening for Biological Properties

The humanized antibodies of the invention may be characterized forspecific binding to FcγRIIB using any immunological or biochemical basedmethod known in the art for characterizing, including quantitating theinteraction of the antibody to FcγRIIB. Specific binding of a humanizedantibody of the invention to FcγRIIB may be determined, for example,using immunological or biochemical based methods including, but notlimited to, an ELISA assay, surface plasmon resonance assays,immunoprecipitation assay, affinity chromatography, fluorescenceactivated cell sorting (FACS), and equilibrium dialysis. Immunoassayswhich can be used to analyze immunospecific binding and cross-reactivityof the antibodies of the invention include, but are not limited to,competitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, protein A immunoassays, to name but afew. Such assays are routine and well known in the art (see, e.g.,Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York, which is incorporated by referenceherein in its entirety).

Humanized antibodies of the invention may be characterized for bindingto FcγRIIB using an in vitro ELISA assay. An exemplary ELISA assay foruse in the methods of the invention may comprise the following: 2.5ng/well of soluble FcγRIIb-Fc fusion protein which is prepared inaccordance with methods disclosed in U.S. Provisional Application No.60/439,709 and U.S. application Ser. No. 10/756,153, both of which areincorporated herein by reference in its entirety, is captured on 96-wellMaxisorp plates by mouse anti-FcγRIIb antibody 3H7 at room temperaturefor 1 hour. A serial of two-fold dilution of conditioned medium of ch2B6or hu2B6Hc/Ch2B6Lc starting from 25 ng/well is added to the each well.The plate is incubated at room temperature for 1 hour, then binding isdetected by HRP conjugated F(ab′)₂ goat anti human IgG F(ab)′2 specificsecondary antibody. After incubation with the secondary antibody forapproximately 45 minutes, the plate is developed using a TMB substrate.After 5 minutes incubation, the reaction is stopped by 1% H₂SO₄. The OD450 nm is read by SOFTmax program. Between each step, the plates arewashed 3 times with PBS/0.1% Tween 20. Plates are blocked by 0.5% BSA inPBS/0.1% Tween 20 for 30 mins at room temperature before adding solubleFcγRIIb-Fc.

Humanized antibodies of the invention may be characterized for bindingto FcγRIIB expressing cells, such as Daudi cells and Rajii cells usingfluorescence activated cell sorting (FACS), using any of the techniquesknown to those skilled in the art. Flow sorters are capable of rapidlyexamining a large number of individual cells (e.g., 10-100 million cellsper hour) (Shapiro et al., Practical Flow Cytometry, 1995). Flowcytometers for sorting and examining biological cells are well known inthe art. Known flow cytometers are described, for example, in U.S. Pat.Nos. 4,347,935; 5,464,581; 5,483,469; 5,602,039; 5,643,796; and6,211,477; the entire contents of which are incorporated by referenceherein. Other known flow cytometers are the FACS Vantage™ systemmanufactured by Becton Dickinson and Company, and the COPAS™ systemmanufactured by Union Biometrica. An exemplary FACS analysis forcharacterizing the humanized antibodies of the invention may comprisethe following: Approximately 10⁶ FcγRIIB expressing cells, e.g., Daudicells and Rajii cells, are washed at least once with a buffer such asPBS. Primary antibodies (e.g., Ch2B6, Hu2B6Hc/ch2B6Lc, human IgG1) arediluted, e.g., into 0.5, 0.1, 0.02 μg/mL in PBS/1% BSA and 100 μl ofdiluted antibodies are transferred to the cells. After 30 minsincubation at 4° C., cells are washed once with 1 ml PBS/1% BSA. PEconjugated F(ab′)₂ fragment of goat anti human IgG Fc specific (JacksonImmunoReseach, Inc.) is used as secondary antibody at 1:1000 dilution.After 30 mins incubation at 4° C., the cells are washed once with 1 mlPBS/1% BSA. Then the cells are resuspended in 500 μl of PBS/1% BSA andsubjected to FACS analysis. Other cell lines that may be used in themethods of the invention include, but are not limited to, CHO-K1(hamster cell line) cells transfected with CD32B; CHO-K1 (hamster cellline) cells transfected with CD32A; 293H (human epithelial cell line)cells transfected with CD32B; 293H (human epithelial cell line) cellstransfected with CD32A; Raji (human Burkitt's lymphoma cell line) cells;Daudi (human Burkitt's lymphoma cell line) cells [Raji and Daudi B celllines express only endogenous CD32B]; THP-1 (human monocytic cell line)cells expressing only endogenous CD32A; U937 (human monocytic cell line)cells expressing endogenous CD32A and CD32B; K526; HL60.

Humanized antibodies of the invention may be further characterized byepitope mapping, so that antibodies may be selected that have thegreatest specificity for FcγRIIB compared to FcγRIIA. Epitope mappingmethods of antibodies are well known in the art and encompassed withinthe methods of the invention. In certain embodiments, FcγRIIB, or afusion protein comprising one or more regions of FcγRIIB, may be used inmapping the epitope of an antibody of the invention. In a specificembodiment, the fusion protein contains the amino acid sequence of aregion of an FcγRIIB fused to the Fc portion of human IgG2. Each fusionprotein may further comprise amino acid substitutions and/orreplacements of certain regions of the receptor with the correspondingregion from a homolog receptor, e.g., FcγRIIA, as shown in Table 2below. pMGX125 and pMGX132 contain the IgG binding site of the FcγRIIBreceptor, the former with the C-terminus of FcγRIIB and the latter withthe C-terminus of FcγRIIA and can be used to differentiate C-terminusbinding. The others have FcγRIIA substitutions in the IgG binding siteand either the FcγIIA or FcγIIB N-terminus. These molecules can helpdetermine the part of the receptor molecule where the antibodies bind.

TABLE 2 List of the fusion proteins that may be used to investigate theepitope of the monoclonal anti-FcγRIIB antibodies. Residues 172to 180 belong to the IgG binding site of FcγRIIA and B. Thespecific amino acids from FcγRIIA sequence are in bold. SEQ SEQ N- ID IDPlasmid Receptor terminus 172-180 NO: C-terminus NO: pMGX125 RIIb IIbKKFSRSDPN 51 APS------SS(IIb) 57 pMGX126 RIIa/b IIa QKFSRLDPN 52APS------SS(IIb) 57 pMGX127 IIa QKFSRLDPT 53 APS------SS(IIb) 57 pMGX128IIb KKFSRLDPT 54 APS------SS(IIb) 57 pMGX129 IIa QKFSHLDPT 55APS------SS(IIb) 57 pMGX130 IIb KKFSHLDPT 56 APS------SS(IIb) 57 pMGX131IIa QKFSRLDPN 52 VPSMGSSS(IIa) 58 pMGX132 IIb KKFSRSDPN 51 VPSMGSSS(IIa)58 pMGX133 RIIa-131R IIa QKFSRLDPT 53 VPSMGSSS(IIa) 58 pMGX134 RIIa-131HIIa QKFSHLDPT 55 VPSMGSSS(IIa) 58 pMGX135 IIb KKFSRLDPT 54 VPSMGSSS(IIa)58 pMGX136 IIb KKFSHLDPT 56 VPSMGSSS(IIa) 58

The fusion proteins may be used in any biochemical assay fordetermination of binding to an anti-FcγRIIB antibody of the invention,e.g., an ELISA. In other embodiments, further confirmation of theepitope specificity may be done by using peptides with specific residuesreplaced with those from the FcγRIIA sequence.

The antibodies of the invention may be characterized for specificbinding to FcγRIIB using any immunological or biochemical based methodknown in the art for characterizing including quantitating, theinteraction of the antibody to FcγRIIB. Specific binding of an antibodyof the invention to FcγRIIB may be determined for example usingimmunological or biochemical based methods including, but not limitedto, an ELISA assay, surface plasmon resonance assays,immunoprecipitation assay, affinity chromatography, and equilibriumdialysis. Immunoassays which can be used to analyze immunospecificbinding and cross-reactivity of the antibodies of the invention include,but are not limited to, competitive and non-competitive assay systemsusing techniques such as western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al., eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).

Humanized antibodies of the invention may also be assayed using anysurface plasmon resonance based assays known in the art forcharacterizing the kinetic parameters of the interaction of the antibodywith FcγRIIB. Any SPR instrument commercially available including, butnot limited to, BIACORE™ Instruments, available from Biacore AB(Uppsala, Sweden); IASYS™ instruments available from Affinity Sensors(Franklin, Mass.); IBIS™ system available from Windsor ScientificLimited (Berks, UK); SPR-CELLIA™ systems available from Nippon Laser andElectronics Lab (Hokkaido, Japan); and SPR Detector SPREETA® availablefrom Texas Instruments (Dallas, Tex.) can be used in the instantinvention. For a review of SPR-based technology, see Mullet et al.,2000, Methods 22: 77-91; Dong et al., 2002, Review in Mol. Biotech., 82:303-23; Fivash et al., 1998, Current Opinion in Biotechnology 9: 97-101;Rich et al., 2000, Current Opinion in Biotechnology 11: 54-61; all ofwhich are incorporated herein by reference in their entirety.Additionally, the methods of the invention contemplate the use of any ofthe SPR instruments and SPR based methods for measuring protein-proteininteractions described in U.S. Pat. Nos. 6,373,577; 6,289,286;5,322,798; 5,341,215; and 6,268,125, all of which are incorporatedherein by reference in their entirety, are contemplated in the methodsof the invention.

Briefly, SPR based assays involve immobilizing a member of a bindingpair on a surface, and monitoring its interaction with the other memberof the binding pair in solution in real time. SPR is based on measuringthe change in refractive index of the solvent near the surface thatoccurs upon complex formation or dissociation. The surface onto whichthe immobilization occur is the sensor chip, which is at the heart ofthe SPR technology; the sensor chip consists of a glass surface coatedwith a thin layer of gold and forms the basis for a range of specializedsurfaces designed to optimize the binding of a molecule to the surface.A variety of sensor chips are commercially available especially from thecompanies listed supra, all of which may be used in the methods of theinvention. Examples of sensor chips include those available from BIAcoreAB, Inc., e.g., Sensor Chip™ CM5™, SA™, NTA™, and HPA™. A molecule ofthe invention may be immobilized onto the surface of a sensor chip usingany of the immobilization methods and chemistries known in the art,including, but not limited to, direct covalent coupling via aminegroups, direct covalent coupling via sulfhydryl groups, biotinattachment to avidin coated surface, aldehyde coupling to carbohydrategroups, and attachment through the histidine tag with NTA chips.

The invention encompasses characterization of the humanized antibodiesproduced by the methods of the invention using certain characterizationassays for identifying the function of the antibodies of the invention,particularly the activity to modulate FcγRIIB signaling. For example,characterization assays of the invention can measure phosphorylation oftyrosine residues in the ITIM motif of FcγRIIB, or measure theinhibition of B cell receptor-generated calcium mobilization. Thecharacterization assays of the invention can be cell-based or cell-freeassays.

It has been well established in the art that in mast cells coaggregationof FcγRIIB with the high affinity IgE receptor, FcεRI, leads toinhibition of antigen-induced degranulation, calcium mobilization, andcytokine production (Metcalfe D. D. et al. 1997, Physiol. Rev. 77:1033;Long E. O. 1999 Annu Rev. Immunol 17: 875). The molecular details ofthis signaling pathway have been recently elucidated (Ott V. L., 2002,J. Immunol. 162(9):4430-9). Once coaggregated with FcεRI, FcγRIIB israpidly phosphorylated on tyrosine in its ITIM motif, and then recruitsSrc Homology-2 containing inositol-5-phosphatase (SHIP), an SH2domain-containing inosital polyphosphate 5-phosphatase, which is in turnphosphorylated and associates with Shc and p62^(dok) (p62^(dok) is theprototype of a family of adaptor molecules, which includes signalingdomains such as an aminoterminal pleckstrin homology domain (PH domain),a PTB domain, and a carboxy terminal region containing PXXP motifs andnumerous phosphorylation sites (Carpino et al., 1997 Cell, 88: 197;Yamanshi et al., 1997, Cell, 88:205)). Each of the foregoing referencesare incorporated herein by reference in its entirety.

The invention encompasses characterizing the anti-FcγRIIB humanizedantibodies of the invention in modulating one or more IgE mediatedresponses. Preferably, cells lines co-expressing the high affinityreceptor for IgE and the low affinity receptor for FcγRIIB will be usedin characterizing the anti-FcγRIIB antibodies of the invention inmodulating IgE mediated responses. In a specific embodiment, cells froma rat basophilic leukemia cell line (RBL-H23; Barsumian E. L. et al.1981 Eur. J. Immunol. 11:317, which is incorporated herein by referencein its entirety) transfected with full length human FcγRIIB will be usedin the methods of the invention. RBL-2H3 is a well characterized ratcell line that has been used extensively to study the signalingmechanisms following IgE-mediated cell activation. When expressed inRBL-2H3 cells and coaggregated with FcεRI, FcγRIIB inhibitsFcεRI-induced calcium mobilization, degranulation, and cytokineproduction (Malbec et al., 1998, J. Immunol. 160:1647; Daeron et al.,1995 J. Clin. Invest. 95:577; Ott et al., 2002 J. of Immunol.168:4430-4439; (each of which is incorporated by reference in itsentirety)).

In some embodiments, the invention encompasses characterizing theanti-FcγRIIB humanized antibodies of the invention for inhibition ofFcεRI induced mast cell activation. For example, cells from a ratbasophilic leukemia cell line (RBL-H23; Barsumian E. L. et al. 1981 Eur.J. Immunol. 11:317; incorporated by reference in its entirety) that havebeen transfected with FcγRIIB are sensitized with IgE and stimulatedeither with F(ab′)₂ fragments of rabbit anti-mouse IgG, to aggregateFcεRI alone, or with whole rabbit anti-mouse IgG to coaggregate FcγRIIBand FcεRI. In this system, indirect modulation of down stream signalingmolecules can be assayed upon addition of antibodies of the invention tothe sensitized and stimulated cells. For example, tyrosinephosphorylation of FcγRIIB and recruitment and phosphorylation of SHIP,activation of MAP kinase family members, including, but not limited toErk1, Erk2, JNK, or p38; and tyrosine phosphorylation of p62^(dok) andits association with SHIP and RasGAP can be assayed.

One exemplary assay for determining the inhibition of FcεRI induced mastcell activation by the antibodies of the invention can comprise of thefollowing: transfecting RBL-H23 cells with human FcγRIIB; sensitizingthe RBL-H23 cells with IgE; stimulating RBL-H23 cells with eitherF(ab′)₂ of rabbit anti-mouse IgG (to aggregate FcεRI alone and elicitFcεRI-mediated signaling, as a control), or stimulating RBL-H23 cellswith whole rabbit anti-mouse IgG to (to coaggregate FcγRIIB and FcεRI,resulting in inhibition of FcεRI-mediated signaling). Cells that havebeen stimulated with whole rabbit anti-mouse IgG antibodies can befurther pre-incubated with the antibodies of the invention. MeasuringFcεRI-dependent activity of cells that have been pre-incubated with theantibodies of the invention and cells that have not been pre-incubatedwith the antibodies of the invention, and comparing levels ofFcεRI-dependent activity in these cells, would indicate a modulation ofFcεRI-dependent activity by the antibodies of the invention.

The exemplary assay described above can be used, for example, toidentify antibodies that block ligand (IgG) binding to FcγRIIB receptorand antagonize FcγRIIB-mediated inhibition of FcεRI signaling bypreventing coaggregating of FcγRIIB and FcεRI. This assay likewiseidentifies antibodies that enhance coaggregation of FcγRIIB and FcεRIand agonize FcγRIIB-mediated inhibition of FcεRI signaling by promotingcoaggregating of FcγRIIB and FcεRI.

In a preferred embodiment, FcεRI-dependent activity is at least one ormore of the following: modulation of downstream signaling molecules,e.g., modulation of phosphorylation state of FcγRIIB, modulation of SHIPrecruitment, modulation of MAP Kinase activity, modulation ofphosphorylation state of SHIP, modulation of SHIP and Shc associationSHIP and Shc, modulation of the phosphorylation state of p62^(dok)modulation of p62^(dok) and SHIP association, modulation of p62^(dok)and RasGAP association, modulation of calcium mobilization, modulationof degranulation, and modulation of cytokine production. In yet anotherpreferred embodiment, FcεRI-dependent activity is serotonin releaseand/or extracellular Ca⁺⁺ influx and/or IgE dependent mast cellactivation. It is known to one skilled in the art that coaggregation ofFcγRIIB and FcεRI stimulates FcγRIIB tyrosine phosphorylation,stimulates recruitment of SHIP, stimulates SHIP tyrosine phosphorylationand association with Shc, and inhibits activation of MAP kinase familymembers including, but not limited to, Erk1, Erk2, JNK, p38. It is alsoknown to those skilled in the art that coaggregation of FcγRIIB andFcεRI stimulates enhanced tyrosine phosphorylation of p62^(dok) and itsassociation with SHIP and RasGAP.

In some embodiments, the anti-FcγRIIB humanized antibodies of theinvention are characterized for their ability to modulate an IgEmediated response by monitoring and/or measuring degranulation of mastcells or basophils, preferably in a cell-based assay. Preferably, mastcells or basophils for use in such assays have been engineered tocontain human FcγRIIB using standard recombinant methods known to oneskilled in the art. In a specific embodiment the anti-FcγRIIB antibodiesof the invention are characterized for their ability to modulate an IgEmediated response in a cell-based β-hexosaminidase (enzyme contained inthe granules) release assay. β-hexosaminidase release from mast cellsand basophils is a primary event in acute allergic and inflammatorycondition (Aketani et al., 2001 Immunol. Lett. 75: 185-9; Aketani etal., 2000 Anal. Chem. 72: 2653-8 (each of which is incorporated byreference in its entirety)). Release of other inflammatory mediatorsincluding, but not limited to, serotonin and histamine may be assayed tomeasure an IgE mediated response in accordance with the methods of theinvention. Although not intending to be bound by a particular mechanismof action, release of granules such as those containing β-hexosaminidasefrom mast cells and basophils is an intracellular calcium concentrationdependent process that is initiated by the cross-linking of FcεRIs withmultivalent antigen.

One exemplary assay for characterizing the anti-FcγRIIB humanizedantibodies of the invention in mediating an IgE mediated response is aβ-hexosaminidase release assay comprising the following: transfectingRBL-H23 cells with human FcγRIIB; sensitizing the cells with mouse IgEalone or with mouse IgE and an anti-FcγRIIB antibody of the invention;stimulating the cells with various concentrations of goat anti-mouseF(ab)₂, preferably in a range from 0.03 μg/mL to 30 μg/mL for about 1hour; collecting the supernatant; lysing the cells; and measuring theβ-hexosaminidase activity released in the supernatant by a colorometricassay, e.g., using p-nitrophenyl N-acetyl-β-D-glucosaminide. Thereleased β-hexosaminidase activity is expressed as a percentage of thereleased activity to the total activity. The released β-hexosaminidaseactivity will be measured and compared in cells treated with antigenalone; IgE alone; IgE and an anti-FcγRIIB antibody of the invention.Although not intending to be bound by a particular mechanism of action,once cells are sensitized with mouse IgE alone and challenged withF(ab)_(z) fragments of a polyclonal goat anti-mouse IgG, aggregation andcross linking of FcεRI occurs since the polyclonal antibody recognizesthe light chain of the murine IgE bound to the FcεRI, which in turnleads to mast cell activation and degranulation. On the other hand, whencells are sensitized with mouse IgE and an anti-FcγRIIB antibody of theinvention and challenged with F(ab)₂ fragments of a polyclonal goatanti-mouse IgG; cross linking of FcεRI and FcγRIIB occurs, resulting ininhibition of FcεRI induced degranulation. In either case, goat antimouse F(ab)₂ induces a dose-dependent β-hexoaminidase release. In someembodiments, the anti-FcγRIIB antibodies bound to the FcγRIIB receptorand cross linked to FcεRI do not affect the activation of the inhibitorypathway, i.e., there is no alteration in the level of degranulation inthe presence of an anti-FcγRIIB antibody. In other embodiments, theanti-FcγRIIB antibodies mediate a stronger activation of the inhibitoryreceptor, FcγRIIB, when bound by the anti-FcγRIIB antibody, allowingeffective cross linking to FcεRI and activation of the inhibitorypathway of homo-aggregated FcγRIIB.

The invention also encompasses characterizing the effect of theanti-FcγRIIB humanized antibodies of the invention on IgE mediated cellresponse using calcium mobilization assays using methodologies known toone skilled in the art. An exemplary calcium mobilization assay maycomprise the following: priming basophils or mast cells with IgE;incubating the cells with a calcium indicator, e.g., Fura 2; stimulatingcells as described supra; and monitoring and/or quantitatingintracellular calcium concentration for example by using flow cytometry.The invention encompasses monitoring and/or quantitating intracellularcalcium concentration by any method known to one skilled in the art.See, e.g., Immunology Letters, 2001, 75:185-9; British J. of Pharm,2002, 136:837-45; J. of Immunology, 168:4430-9 and J. of Cell Biol.,153(2):339-49; all of which are incorporated herein by reference.

In preferred embodiments, anti-FcγRIIB humanized antibodies of theinvention inhibit IgE mediated cell activation. In other embodiments,the anti-FcγRIIB antibodies of the invention block the inhibitorypathways regulated by FcγRIIB or block the ligand binding site onFcγRIIB and thus enhance immune response.

The ability to study human mast cells has been limited by the absence ofsuitable long term human mast cell cultures. Recently two novel stemcell factor dependent human mast cell lines, designated LAD 1 and LAD2,were established from bone marrow aspirates from a patient with mastcell sarcoma/leukemia (Kirshenbaum et al., 2003, Leukemia research,27:677-82, which is incorporated herein by reference in its entirety.).Both cell lines have been described to express FcεRI and several humanmast cell markers. The invention encompasses using LAD 1 and 2 cells inthe methods of the invention for assessing the effect of the humanizedantibodies of the invention on IgE mediated responses. In a specificembodiment, cell-based β-hexosaminidase release assays such as thosedescribed supra may be used in LAD cells to determine any modulation ofthe IgE-mediated response by the humanized anti-FcγRIIB antibodies ofthe invention. In an exemplary assay, human mast cells, e.g., LAD 1, areprimed with chimaeric human IgE anti-nitrophenol (NP) and challengedwith BSA-NP, the polyvalent antigen, and cell degranulation is monitoredby measuring the β-hexosaminidase released in the supernatant(Kirshenbaum et al., 2003, Leukemia research, 27:677-682, which isincorporated herein by reference in its entirety).

In some embodiments, if human mast cells have a low expression ofendogenous FcγRIIB, as determined using standard methods known in theart, e.g., FACS staining, it may be difficult to monitor and/or detectdifferences in the activation of the inhibitory pathway mediated by theanti-FcγRIIB antibodies of the invention. The invention thus encompassesalternative methods, whereby the FcγRIIB expression may be upregulatedusing cytokines and particular growth conditions. FcγRIIB has beendescribed to be highly up-regulated in human monocyte cell lines, e.g.,THP1 and U937, (Tridandapani et al., 2002, J. Biol. Chem., 277(7):5082-5089) and in primary human monocytes (Pricop et al., 2001, J. ofImmunol., 166: 531-537) by IL4. Differentiation of U937 cells withdibutyryl cyclic AMP has been described to increase expression of FcγRII(Cameron et al., 2002 Immunology Letters 83, 171-179). Thus, theendogenous FcγRIIB expression in human mast cells for use in the methodsof the invention may be up-regulated using cytokines, e.g., IL-4, IL-13,in order to enhance sensitivity of detection. Each of the foregoingreferences is hereby incorporated by reference in its entirety.

The invention also encompasses characterizing the humanized anti-FcγRIIBantibodies of the invention for inhibition of B-cell receptor(BCR)-mediated signaling. BCR-mediated signaling can include at leastone or more down stream biological responses, such as activation andproliferation of B cells, antibody production, etc. Coaggregation ofFcγRIIB and BCR leads to inhibition of cell cycle progression andcellular survival. Further, coaggregation of FcγRIIB and BCR leads toinhibition of BCR-mediated signaling.

Specifically, BCR-mediated signaling comprises at least one or more ofthe following: modulation of down stream signaling molecules (e.g.,phosphorylation state of FcγRIIB, SHIP recruitment, localization of Btkand/or PLCγ, MAP kinase activity, recruitment of Akt (anti-apoptoticsignal), calcium mobilization, cell cycle progression, and cellproliferation.

Although numerous effector functions of FcγRIIB-mediated inhibition ofBCR signaling are mediated through SHIP, recently it has beendemonstrated that lipopolysaccharide (LPS)-activated B cells from SHIPdeficient mice exhibit significant FcγRIIB-mediated inhibition ofcalcium mobilization, Ins(1,4,5)P₃ production, and Erk and Aktphosphorylation (Brauweiler A. et al., 2001, Journal of Immunology,167(1): 204-211; incorporated by reference in its entirety).Accordingly, ex vivo B cells from SHIP deficient mice can be used tocharacterize the antibodies of the invention. One exemplary assay fordetermining FcγRIIB-mediated inhibition of BCR signaling by theantibodies of the invention can comprise the following: isolatingsplenic B cells from SHIP deficient mice, activating said cells withlipopolysachharide, and stimulating said cells with either F(ab′)₂anti-IgM to aggregate BCR or with anti-IgM to coaagregate BCR withFcγRIIB. Cells that have been stimulated with intact anti-IgM tocoaggregate BCR with FcγRIIB can be further pre-incubated with theantibodies of the invention. FcγRIIB-dependent activity of cells can bemeasured by standard techniques known in the art and used for, e.g.,comparing the level of FcγRIIB-dependent activity in cells that havebeen pre-incubated with the antibodies of the invention and cells thathave not been pre-incubated, and comparing the levels would indicate amodulation of FcγRIIB-dependent activity by the antibodies of theinvention.

Measuring FcγRIIB-dependent activity can include, for example, measuringintracellular calcium mobilization by flow cytometry, measuringphosphorylation of Akt and/or Erk, measuring BCR-mediated accumulationof PI(3,4,5)P₃, or measuring FcγRIIB-mediated proliferation B cells.

The assays can be used, for example, to identify antibodies thatmodulate FcγRIIB-mediated inhibition of BCR signaling by blocking theligand (IgG) binding site to FcγRIIB receptor and antagonizingFcγRIIB-mediated inhibition of BCR signaling by preventing coaggregationof FcγRIIB and BCR. The assays can also be used to identify antibodiesthat enhance coaggregation of FcγRIIB and BCR and agonizeFcγRIIB-mediated inhibition of BCR signaling.

The invention relates to characterizing the humanized anti-FcγRIIBantibodies of the invention for FcγRII-mediated signaling in humanmonocytes/macrophages. Coaggregation of FcγRIIB with a receptor bearingthe immunoreceptor tyrosine-based activation motif (ITAM) acts todown-regulate FcγR-mediated phagocytosis using SHIP as its effector(Tridandapani et al. 2002, J. Biol. Chem. 277(7):5082-9; incorporated byreference in its entirety). Coaggregation of FcγRIIA with FcγRIIBresults in rapid phosphorylation of the tyrosine residue on FcγRIIB'sITIM motif, leading to an enhancement in phosphorylation of SHIP,association of SHIP with Shc, and phosphorylation of proteins having themolecular weight of 120 and 60-65 kDa. In addition, coaggregation ofFcγRIIA with FcγRIIB results in down-regulation of phosphorylation ofAkt, which is a serine-threonine kinase that is involved in cellularregulation and serves to suppress apoptosis.

The invention further encompasses characterizing the humanizedanti-FcγRIIB antibodies of the invention for their inhibition ofFcγR-mediated phagocytosis in human monocytes/macrophages. For example,cells from a human monocytic cell line, THP-1 can be stimulated eitherwith Fab fragments of mouse monoclonal antibody IV.3 against FcγRIIA(Medarex, Inc.) and goat anti-mouse antibody (to aggregate FcγRIIAalone), or with whole IV.3 mouse monoclonal antibody and goat anti-mouseantibody (to coaggregate FcγRIIA and FcγRIIB). In this system,modulation of down stream signaling molecules, such as tyrosinephosphorylation of FcγRIIB, phosphorylation of SHIP, association of SHIPwith Shc, phosphorylation of Akt, and phosphorylation of proteins havingthe molecular weight of 120 and 60-65 kDa can be assayed upon additionof antibodies of the invention to the stimulated cells. In addition,FcγRIIB-dependent phagocytic efficiency of the monocyte cell line can bedirectly measured in the presence and absence of the antibodies of theinvention.

Another exemplary assay for determining inhibition of FcγR-mediatedphagocytosis in human monocytes/macrophages by the antibodies of theinvention can comprise the following: stimulating THP-1 cells witheither Fab of IV.3 mouse anti-FcγRIIA antibody and goat anti-mouseantibody (to aggregate FcγRIIA alone and elicit FcγRIIA-mediatedsignaling); or with mouse anti-FcγRII antibody and goat anti-mouseantibody (to coaggregate FcγRIIA and FcγRIIB and inhibitingFcγRIIA-mediated signaling). Cells that have been stimulated with mouseanti-FcγRII antibody and goat anti-mouse antibody can be furtherpre-incubated with the antibodies of the invention. MeasuringFcγRIIA-dependent activity of stimulated cells that have beenpre-incubated with antibodies of the invention and cells that have notbeen pre-incubated with the antibodies of the invention and comparinglevels of FcγRIIA-dependent activity in these cells would indicate amodulation of FcγRIIA-dependent activity by the antibodies of theinvention.

The exemplary assay described can be used, for example, to identifyantibodies that block ligand binding of FcγRIIB receptor and antagonizeFcγRIIB-mediated inhibition of FcγRIIA signaling by preventingcoaggregation of FcγRIIB and FcγRIIA. This assay likewise identifiesantibodies that enhance coaggregation of FcγRIIB and FcγRIIA and agonizeFcγRIIB-mediated inhibition of FcγRIIA signaling.

In another embodiment of the invention, the invention relates tocharacterizing the function of the humanized antibodies of the inventionby measuring the ability of THP-1 cells to phagocytose fluoresceinatedIgG-opsonized sheep red blood cells (SRBC) by methods previouslydescribed (Tridandapani et al., 2000, J. Biol. Chem. 275: 20480-7;incorporated by reference in its entirety). For example, an exemplaryassay for measuring phagocytosis comprises: treating THP-1 cells withthe antibodies of the invention or with a control antibody that does notbind to FcγRII, comparing the activity levels of said cells, wherein adifference in the activities of the cells (e.g., rosetting activity (thenumber of THP-1 cells binding IgG-coated SRBC), adherence activity (thetotal number of SRBC bound to THP-1 cells), and phagocytic rate) wouldindicate a modulation of FcγRIIA-dependent activity by the antibodies ofthe invention. This assay can be used to identify, for example,antibodies that block ligand binding of FcγRIIB receptor and antagonizeFcγRIIB-mediated inhibition of phagocytosis. This assay can alsoidentify antibodies that enhance FcγRIIB-mediated inhibition of FcγRIIAsignaling.

In a preferred embodiment, the humanized antibodies of the inventionmodulate FcγRIIB-dependent activity in human monocytes/macrophages in atleast one or more of the following ways: modulation of downstreamsignaling molecules (e.g., modulation of phosphorylation state ofFcγRIIB, modulation of SHIP phosphorylation, modulation of SHIP and Shcassociation, modulation of phosphorylation of Akt, modulation ofphosphorylation of additional proteins around 120 and 60-65 kDa) andmodulation of phagocytosis.

The invention encompasses characterization of the humanized antibodiesof the invention using assays known to those skilled in the art foridentifying the effect of the antibodies on effector cell function oftherapeutic antibodies, e.g., their ability to enhance tumor-specificADCC activity of therapeutic antibodies. Therapeutic antibodies that maybe used in accordance with the methods of the invention include, but arenot limited to, anti-tumor antibodies, anti-viral antibodies,anti-microbial antibodies (e.g., bacterial and unicellular parasites),examples of which are disclosed herein (Section 5.3.6). In particular,the invention encompasses characterizing the antibodies of the inventionfor their effect on FcγR-mediated effector cell function of therapeuticantibodies, e.g., tumor-specific monoclonal antibodies. Examples ofeffector cell functions that can be assayed in accordance with theinvention, include, but are not limited to, antibody-dependent cellmediated cytotoxicity, phagocytosis, opsonization, opsonophagocytosis,Clq binding, and complement dependent cell mediated cytotoxicity. Anycell-based or cell free assay known to those skilled in the art fordetermining effector cell function activity can be used (for effectorcell assays, see Perussia et al., 2000, Methods Mol. Biol. 121: 179-92;Baggiolini et al., 1998 Experientia, 44(10): 841-8; Lehmann et al., 2000J. Immunol. Methods, 243(1-2): 229-42; Brown E J. 1994, Methods CellBiol., 45: 147-64; Munn et al., 1990 J. Exp. Med., 172: 231-237,Abdul-Majid et al., 2002 Scand. J. Immunol. 55: 70-81; Ding et al.,1998, Immunity 8:403-411, each of which is incorporated by referenceherein in its entirety).

Antibodies of the invention can be assayed for their effect onFcγR-mediated ADCC activity of therapeutic antibodies in effector cells,e.g., natural killer cells, using any of the standard methods known tothose skilled in the art (see e.g., Perussia et al., 2000, Methods Mol.Biol. 121: 179-92). “Antibody-dependent cell-mediated cytotoxicity” and“ADCC”, as used herein, carry their ordinary and customary meaning inthe art and refer to an in vitro cell-mediated reaction in whichnonspecific cytotoxic cells that express FcγRs (e.g., monocytic cellssuch as Natural Killer (NK) cells and macrophages) recognize boundantibody on a target cell and subsequently cause lysis of the targetcell. In principle, any effector cell with an activating FcγR can betriggered to mediate ADCC. The primary cells for mediating ADCC are NKcells which express only FcγRIII, whereas monocytes, depending on theirstate of activation, localization, or differentiation, can expressFcγRI, FcγRII, and FcγRIII. For a review of FcγR expression onhematopoietic cells, see, e.g., Ravetch et al., 1991, Annu. Rev.Immunol., 9:457-92, which is incorporated herein by reference in itsentirety.

Effector cells are leukocytes which express one or more FcγRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Effector cells that may beused in the methods of the invention include, but are not limited to,peripheral blood mononuclear cells (PBMC), natural killer (NK) cells,monocytes, and neutrophils; with PBMCs and NK cells being preferred. Theeffector cells may be isolated from a native source thereof, e.g., fromblood or PBMCs as described herein. Preferably, the effector cells usedin the ADCC assays of the invention are peripheral blood mononuclearcells (PBMC) that are preferably purified from normal human blood, usingstandard methods known to one skilled in the art, e.g., usingFicoll-Paque density gradient centrifugation. For example, PBMCs may beisolated by layering whole blood onto Ficoll-Hypaque and spinning thecells at 500 g, at room temperature for 30 minutes. The leukocyte layercan be harvested as effector cells. Other effector cells that may beused in the ADCC assays of the invention include, but are not limitedto, monocyte-derived macrophages (MDMs). MDMs that are used as effectorcells in the methods of the invention are preferably obtained as frozenstocks or used fresh (e.g., from Advanced Biotechnologies, Md.). In mostpreferred embodiments, elutriated human monocytes are used as effectorcells in the methods of the invention. Elutriated human monocytesexpress activating receptors, FcγRIIIA and FcγRIIA and the inhibitoryreceptor, FcγRIIB. Human monocytes are commercially available and may beobtained as frozen stocks, thawed in basal medium containing 10% humanAB serum or in basal medium with human serum containing cytokines.Levels of expression of FcγRs in the cells may be directly determined;e.g. using FACS analysis. Alternatively, cells may also be allowed tomature to macrophages in culture. The level of FcγRIIB expression may beincreased in macrophages. Antibodies that may be used in determining theexpression level of FcγRs include but are not limited to anti-humanFcγRIIA antibodies, e.g., IV.3-FITC; anti-FcγRI antibodies, e.g., 32.2FITC; and anti-FcγRIIIA antibodies, e.g., 3G8-PE.

Target cells used in the ADCC assays of the invention include, but arenot limited to, breast cancer cell lines, e.g., SK-BR-3 with ATCCaccession number HTB-30 (see, e.g., Tremp et al., 1976, Cancer Res.33-41); B-lymphocytes; cells derived from Burkitts lymphoma, e.g., Rajicells with ATCC accession number CCL-86 (see, e.g., Epstein et al.,1965, J. Natl. Cancer Inst. 34: 231-240), Daudi cells with ATCCaccession number CCL-213 (see, e.g., Klein et al., 1968, Cancer Res. 28:1300-10); ovarian carcinoma cell lines, e.g., OVCAR-3 with ATCCaccession number HTB-161 (see, e.g., Hamilton, Young et al., 1983),SK-OV-3, PA-1, CAOV3, OV-90, and IGROV-1 (available from the NCIrepository; Benard et al., 1985, Cancer Research, 45:4970-9; which isincorporated herein by reference in its entirety. The target cells mustbe recognized by the antigen binding site of the antibody to be assayed.The target cells for use in the methods of the invention may have low,medium, or high expression level of a cancer antigen. The expressionlevels of the cancer antigen may be determined using common methodsknown to one skilled in the art, e.g., FACS analysis. For example, theinvention encompasses the use of ovarian cancer cells such as IGROV-1,wherein Her2/neu is expressed at different levels, or OV-CAR-3 (ATCCAssession Number HTB-161; characterized by a lower expression ofHer2/neu than SK-BR-3, the breast carcinoma cell line). Other ovariancarcinoma cell lines that may be used as target cells in the methods ofthe invention include OVCAR-8 (Hamilton et al., 1983, Cancer Res.43:5379-89, which is incorporated herein by reference in its entirety);SK-OV-3 (ATCC Accession Number HTB-77); Caov-3 (ATCC Accession NumberHTB-75); PA-1 (ATCC Accession Number CRL-1572); OV-90 (ATCC AccessionNumber CRL-11732); and OVCAR-4. Other breast cancer cell lines that maybe used in the methods of the invention include BT-549 (ATCC AccessionNumber HTB-122), MCF7 (ATCC Accession Number HTB-22), and Hs578T (ATCCAccession Number HTB-126), all of which are available from the NCIrepository and ATCC and which are incorporated herein by reference.Other cell lines that may be used in the methods of the inventioninclude, but are not limited to, CCRF-CEM (leukemia); HL-60 (TB,leukemia); MOLT-4 (leukemia); RPMI-8226 (leukemia); SR (leukemia); A549(Non-small cell lung); EKVX (Non-small cell lung); HOP-62 (Non-smallcell lung); HOP-92 (Non-small cell lung); NC1-H226 (Non-small celllung); NC1-H23 (Non-small cell lung); NC1-H322M (Non-small cell lung);NC1-H460 (Non-small cell lung); NC1-H522 (Non-small cell lung); COLO 205(Colon); HCC-2998 (Colon); HCT-116 (Colon); HCT-15 (Colon); HT29(Colon); KM12 (Colon); SW-620 (Colon); SF-268 (CNS); SF-295 (CNS);SF-539 (CNS); SNB-19 (CNS); SNB-75 (CNS); U251 (CNS); LOX IMV1(Melanoma); MALME-3M (Melanoma); M14 (Melanoma); SK-MEL-2 (Melanoma);SK-MEL-28 (Melanoma); SK-MEL-5 (Melanoma); UACC-257 (Melanoma); UACC-62(Melanoma); IGR-OV1 (Ovarian); OVCAR-3,4,5,8 (Ovarian); SK-OV-3(Ovarian); 786-0 (Renal); A498 (Renal); ACHN (Renal); CAK1-1 (Renal);SN12C(Renal); TK-10 (Renal); UO-31 (Renal); PC-3C (Prostate); DU-145(Prostate); NC1/ADR-RES (Breast); MDA-MB-231/ATCC (Breast); MDA-MB-435(Breast); DMS 114 (Small-cell lung); and SHP-77 (Small-cell lung); allof which are available from the NC1 and which are incorporated herein byreference.

An exemplary assay for determining the effect of the antibodies of theinvention on the ADCC activity of therapeutic antibodies is based on a⁵¹Cr release assay comprising: labeling target cells with [⁵¹Cr]Na₂CrO₄(this cell-membrane permeable molecule is commonly used for labelingsince it binds cytoplasmic proteins and although spontaneously releasedfrom the cells with slow kinetics, it is released massively followingtarget cell lysis); preferably, the target cells express one or moretumor antigens, osponizing the target cells with one or more antibodiesthat immunospecifically bind the tumor antigens expressed on the cellsurface of the target cells, in the presence and absence of an antibodyof the invention, e.g., 2B6, 3H7, combining the opsonized radiolabeledtarget cells with effector cells in a microtitre plate at an appropriateratio of target cells to effector cells; incubating the mixture of cellspreferably for 16-18 hours, preferably at 37° C.; collectingsupernatants; and analyzing the radioactivity in the supernatantsamples. The cytotoxicity of the therapeutic antibodies in the presenceand absence of the antibodies of the invention can then be determined,for example using the following formula: Percent specificlysis=(Experimental lysis−antibody−independent lysis/maximallysis−antibody independent lysis)×100%. A graph can be generated byvarying either the target: effector cell ratio or antibodyconcentration.

In yet another embodiment, the antibodies of the invention arecharacterized for antibody dependent cellular cytotoxicity (ADCC) inaccordance with the method described earlier; see, e.g., Ding et al.,Immunity, 1998, 8:403-11, which is incorporated herein by reference inits entirety.

In some embodiments, the invention encompasses characterizing thefunction of the antibodies of the invention in enhancing ADCC activityof therapeutic antibodies in an in vitro based assay and/or in an animalmodel.

In a specific embodiment, the invention encompasses determining thefunction of the humanized antibodies of the invention in enhancing tumorspecific ADCC using an ovarian cancer model and/or breast cancer model.

Preferably, the ADCC assays of the invention are done using more thanone cancer cell line, characterized by the expression of at least onecancer antigen, wherein the expression level of the cancer antigen isvaried among the cancer cell lines used. Although not intending to bebound by a particular mechanism of action, performing ADCC assays inmore than one cell line wherein the expression level of the cancerantigen is varied, will allow determination of stringency of tumorclearance of the antibodies of the invention. In one embodiment, theADCC assays of the invention are done using cancer cell lines withdifferent levels of expression of a cancer antigen.

In an exemplary assay, OVCAR3, an ovarian carcinoma cell line, can serveas the tumor target expressing the tumor antigens, Her2/neu and TAG-72;human monocytes that express the activating FcγRIIIA and FcγRIIA andinhibitory FcγRIIB, can be used as effectors; and tumor specific murineantibodies, ch4D5 and chCC49, can be used as the tumor specificantibodies. OVCAR-3 cells are available from ATCC (Accession NumberHTB-161). Preferably, OVCAR-3 cells are propagated in mediumsupplemented with 0.01 mg/ml bovine insulin. 5×10⁶ viable OVCAR-3 cellsmay be injected subcutaneously (s.c) into age and weight matched nudeathymic mice with MATRIGEL® (Becton Dickinson). The estimated weight ofthe tumor can be calculated by the formula: length-(width)/2, andpreferably does not exceed 3 grams. Anchorage-dependent tumor can beisolated after 6-8 weeks, and the cells can be dissociated by adding 1μg of Collagenase (Sigma) per gram of tumor and a 5 mg/mL RNase, passedthrough a cell strainer and nylon mesh to isolate cells. Cells can thenbe frozen for long-term storage for s.c. injection for establishment ofthe xenograft model.

Hybridomas secreting CC49 and 4D5 antibodies are available with ATCCAccession Numbers HB-9459 and CRL-3D463 and the heavy chain and lightchain nucleotide sequences are in the public domain (see, e.g., Murrayet al., 1994 Cancer 73 (35):1057-66, Yamamoto et al., 1986 Nature,319:230-4; both of which are incorporated herein by reference in theirentirety). Preferably, the 4D5 and CC49 antibodies are chimerized usingstandard methods known to one skilled in the art so that the human Fcsequence, e.g., human constant region of IgG1, is grafted onto thevariable region of the murine antibodies in order to provide theeffector function. The chimeric 4D5 and CC49 antibodies bind via theirvariable region to the target cell lines and via their Fc region toFcγRs expressed on human effector cells. CC49 is directed to TAG-72, ahigh molecular weight mucin that is highly expressed on manyadenocarcinoma cells and ovarian carcinoma (Lottich et al., 1985 BreastCancer Res. Treat. 6(1):49-56; Mansi et al., 1989 Int. J. Rad. Appl.Instrum B. 16(2):127-35; Colcher et al., 1991 Int. J. Rad. Appl. InstrumB. 18:395-41; all of which are incorporated herein by reference in theirentirety). 4D5 is directed to human epidermal growth factor receptor 2(Carter et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 4285-9, which isincorporated herein by reference). Antibodies of the invention can thenbe utilized to investigate the enhancement of ADCC activity of the tumorspecific antibodies, by blocking the inhibitory FcγRIIB. Although notintending to be bound by a particular mechanism of action, uponactivation of effector cells that express at least one activating FcγR,e.g., FcγRIIA, the expression of the inhibitory receptor (FcγRIIB) isenhanced and this limits the clearance of tumors as the ADCC activity ofFcγRIIA is suppressed. However, antibodies of the invention can serve asa blocking antibody, i.e., an antibody that will prevent the inhibitorysignal from being activated and thus, the activation signal, e.g., ADCCactivity, will be sustained for a longer period and may result in potenttumor clearance.

Preferably, the humanized antibodies of the invention for use inenhancement of ADCC activity have been modified to comprise at least oneamino acid modification so that binding of their Fc region to FcγR hasbeen diminished, most preferably abolished. In some embodiments, theantibodies of the invention have been modified to comprise at least oneamino acid modification which reduces the binding of the constant domainto an activating FcγR, e.g., FcγRIIIA, FcγRIIA, as compared to a wildtype antibody of the invention while retaining maximal FcγRIIB blockingactivity. Antibodies of the invention may be modified in accordance withany method known to one skilled in the art or disclosed herein. Anyamino acid modification which is known to disrupt effector function maybe used in accordance with the methods of the invention such as thosedisclosed in International Publication Nos. WO 04/063351, WO 04/029207,WO 04/029092, WO 04/028564, WO 99/58572, WO 99/51642, WO 98/23289, WO89/07142, WO 88/07089; U.S. Patent Application Publication Nos.2005/0037000; and 2005/0064514 and U.S. Pat. Nos. 5,843,597 and5,642,821, each of which is incorporated herein by reference in theirentirety. The invention also encompasses any of the mutations disclosedin U.S. patent application Ser. Nos. 10/902,588 (filed Jul. 28, 2004)and 11/271,140 (filed Nov. 10, 2005), and U.S. Provisional ApplicationNos. 60/707,419; and 60/781,564 filed on Aug. 10, 2005, and Mar. 10,2006, respectively, each of which are incorporated herein by referencein their entireties. In some embodiments, antibodies of the inventionare modified so that position 265 is modified, e.g., position 265 issubstituted with alanine. In preferred embodiments, the murine constantregion of an antibody of the invention is swapped with the correspondinghuman constant region comprising a substitution of the amino acid atposition 265 with alanine, so that the effector function is abolishedwhile FcγRIIB blocking activity is maintained. A single amino acidchange at position 265 of IgG1 heavy chain has been shown tosignificantly reduce binding to FcγR based on ELISA assays, and hasresulted in tumor mass reduction (Shields et al., 2001, J. Biol. Chem.,276:6591-6604, which is incorporated herein by reference in itsentirety). In other embodiments, antibodies of the invention aremodified so that position 297 is modified, e.g., position 297 issubstituted with glutamine, so that the N-linked glycosylation site iseliminated (see, e.g., Jefferies et al., 1995, Immunol. lett 44:111-7;Lund et al., 1996, J. Immunol., 157:4963-69; Wright et al., 1994, J.Exp. Med. 180:1087-96; White et al., 1997; J. Immunol. 158:426-35; allof which are incorporated herein by reference in their entireties.Modification at this site has been reported to abolish all interactionwith FcγRs. In preferred embodiments, the murine constant region of anantibody of the invention is swapped with the corresponding humanconstant region comprising a substitution of the amino acid at position265 and/or 297, so that the effector function is abolished while FcγRIIBblocking activity is maintained.

An exemplary assay for determining the ADCC activity of the tumorspecific antibodies in the presence and absence of the antibodies of theinvention is a non-radioactive europium based fluorescent assay (BATDA®,Perkin Elmer) and may comprise the following: labeling the targets cellswith an acteoxylmethyl ester of fluorescence-enhancing ester that formsa hydrophilic ligand (TDA) with the membrane of cells by hydrolysis ofthe esters (this complex is unable to leave the cell and is releasedonly upon lysis of the cell by the effectors); adding the labeledtargets to the effector cells in presence of anti-tumor antibodies andan antibody of the invention; and incubating the mixture of the targetand effector cells for 6 to 16 hours, preferably at 37° C. The extent ofADCC activity can be assayed by measuring the amount of ligand that isreleased and interacts with europium (DELFIA® reagent; PerkinElmer). Theligand and the europium form a very stable and highly fluorescentchelate (EuTDA) and the measured fluorescence is directly proportionalto the number of cells lysed. Percent specific lysis can be calculatedusing the formula: (Experimental lysis−antibody−independentlysis/maximal lysis antibody−independent lysis×100%).

In some embodiments, if the sensitivity of the fluorescence-based ADCCassay is too low to detect ADCC activity of the therapeutic antibodies,the invention encompasses using radioactive-based ADCC assays, such as⁵¹Cr release assay. Radioactive-based assays may be done instead of, orin combination with, fluorescent-based ADCC assays.

An exemplary ⁵¹Cr release assay for characterizing the antibodies of theinvention can comprise the following: labeling 1-2×10⁶ target cells suchas OVCAR-3 cells with ⁵¹Cr; opsonizing the target cells with antibodies4D5 and CC49 in the presence and absence of an antibody of the inventionand adding 5×10³ cells to 96 well plate (preferably 4D5 and CC49 are ata concentration varying from 1-15 μg/mL); adding the opsonized targetcells to monocyte-derived macrophages (MDM) (effector cells), preferablyat a ratio varying from 10:1 to 100:1; incubating the mixture of cellsfor 16-18 hours at 37° C.; collecting supernatants; and analyzing theradioactivity in the supernatant. The cytotoxicity of 4D5 and CC49 inthe presence and absence of an antibody of the invention can then bedetermined, for example, using the following formula percent specificlysis=(experimental lysis−antibody independent lysis/maximallysis−antibody independent lysis)×100%.

In some embodiments, the in vivo activity of the FcγRIIB humanizedantibodies of the invention is determined in xenograft human tumormodels. Tumors may be established using any of the cancer cell linesdescribed supra. In some embodiments, the tumors will be establishedwith two cancer cell lines, wherein the first cancer cell line ischaracterized by a low expression of a cancer antigen and a secondcancer cell line, wherein the second cancer cell line is characterizedby a high expression of the same cancer antigen. Tumor clearance maythen be determined using methods known to one skilled in the art, usingan anti-tumor antibody which immunospecifically binds the cancer antigenon the first and second cancer cell line, and an appropriate mousemodel, e.g., a Balb/c nude mouse model (e.g., Jackson Laboratories,Taconic), with adoptively transferred human monocytes and MDMs aseffector cells. Any of the antibodies described supra may then be testedin this animal model to evaluate the role of anti-FcγRIIB antibody ofthe invention in tumor clearance. Mice that may be used in the inventioninclude for example FcγRIII −/− (where FcγRIIIA is knocked out); Fcγ−/−nude mice (where FcγRI and FcγRIIIA are knocked out); or human FcγRIIBknock in mice or a transgenic knock-in mice, where mousefcgr2 and fcgr3loci on chromosome 1 are inactivated and the mice express human FcγRIIA,human FcγRIIA human FcγRIIB, human FcγRIIC, human FcγRIIIA, and humanFcγRIIIB

An exemplary method for testing the in vivo activity of an antibody ofthe invention may comprise the following: establishing a xenograftmurine model using a cancer cell line characterized by the expression ofa cancer antigen and determining the effect of an antibody of theinvention on an antibody specific for the cancer antigen expressed inthe cancer cell line in mediating tumor clearance. Preferably, the invivo activity is tested parallel using two cancer cell lines, whereinthe first cancer cell line is characterized by a first cancer antigenexpressed at low levels and a second cancer cell line, characterized bythe same cancer antigen expressed at a higher level relative to thefirst cancer cell line. These experiments will thus increase thestringency of the evaluation of the role of an antibody of the inventionin tumor clearance. For example, tumors may be established with theIGROV-1 cell line and the effect of an anti-FcγRIIB antibody of theinvention in tumor clearance of a Her2/neu specific antibody may beassessed. In order to establish the xenograft tumor models, 5×10⁶ viablecells, e.g., IGROV-1, SKBR3, may be injected, e.g., s.c. into mice,e.g., 8 age and weight matched femal nude athymic mice using, forexample, MATRIGEL® (Becton Dickinson). The estimated weight of the tumormay be determined by the formula: length×(width)/2; and preferably doesnot exceed 3 grams. Injection of IGROV-1 cells s.c. gives rise to fastgrowing tumors while the i.p. route induces a peritoneal carcinomatosiswhich kills mice in 2 months (Benard et al., 1985, Cancer Res.45:4970-9; incorporated by reference in its entirety). Since the IGROV-1cells form tumors within 5 weeks, at day 1 after tumor cell injection,monocytes as effectors are co-injected i.p. along with a therapeuticantibody specific for Her2/neu, e.g., Ch4D5, and an antibody of theinvention; e.g. chimeric 2B6 or 3H7 as described supra. Preferably, theantibodies are injected at 4 μg each per gram of mouse body weight(mbw). The initial injection will be followed by weekly injections ofantibodies for 4-6 weeks thereafter at 2 μg/wk. Human effector cellswill be replenished once in 2 weeks. A group of mice will receive notherapeutic antibody but will be injected with a chimeric 4D5 comprisinga N297A mutation and human IgG1 as isotype control antibodies for theanti-tumor and anti-FcγRIIB antibodies, respectively. Mice may be placedin groups of 4 and monitored three times weekly.

Table 3 below is an exemplary setup for tumor clearance studies inaccordance with the invention. As shown in Table 3, six groups of 8 miceeach will be needed for testing the role of an antibody of the inventionin tumor clearance, wherein one target and effector cell combination isused and wherein two different combinations of the antibodyconcentration are used. In group A, only tumor cells are injected; ingroup B, tumor cells and monocytes are injected; in group C, tumorcells, monocytes, an anti-tumor antibody (ch4D5) are injected; in groupD, tumor cells, monocytes, anti-tumor antibody, and an anti-FcγRIIantibody are injected; in group E, tumor cells, monocytes and ananti-FcγRIIB antibody are injected; in group F, tumor cells, monocytes,Ch4D5 (N297Q), and human IgG1 are injected. It will be appreciated byone skilled in the art that various antibody concentrations of variousantibody combinations may be tested in the tumor models described.Preferably, studies using a breast cancer cell line, e.g., SKBR3, iscarried out in parallel to the above-described experiment.

TABLE 3 EXEMPLARY EXPERIMENTAL SET UP IN MICE ch4D5 at ch4D5 N297Q atch2B6 N297Q at Human IgG1 8 mice/ Tumor cell Monocytes 4 μg/gm of 4μg/gm of 4 μg/gm of 4 μg/gm of group s.c day 0 i.p at day 1 mbw day 1i.p mbw day 1 i.p mbw day 1 i.p mbw day 1 i.p A + − − − − − B + + − − −− C + + + − − − D + + + − + − E + + − − + − F + + − + − +

The endpoint of the xenograft tumor models is determined based on thesize of the tumors, weight of mice, survival time and histochemical andhistopathological examination of the cancer, using methods known to oneskilled in the art. Each of the groups of mice in Table 3 will beevaluated. Mice are preferably monitored three times a week. Criteriafor tumor growth may be abdominal distention, presence of palpable massin the peritoneal cavity. Preferably estimates of tumor weight versusdays after inoculation will be calculated. A comparison of theaforementioned criteria of mice in Group D compared to those in othergroups will define the role of an antibody of the invention inenhancement of tumor clearance. Preferably, antibody-treated animalswill be under observation for an additional 2 months after the controlgroup.

In alternative embodiments, human FcγRIIB “knock in” mice expressinghuman FcγRIIB on murine effector cells may be used in establishing thein vivo activity of the antibodies of the invention, rather thanadoptively transferring effector cells. Founder mice expressing thehuman FcγRIIB may be generated by “knocking in” the human FcγRIIB ontothe mouse FcγRIIB locus. The founders can then be back-crossed onto thenude background and will express the human FcγRIIB receptor. Theresulting murine effector cells will express endogenous activating FcγRIand FcγRIIIA and inhibitory human FcγRIIB receptors.

The in vivo activity of the humanized antibodies of the invention may befurther tested in a xenograft murine model with human primary tumorderived cells, such as human primary ovarian and breast carcinomaderived cells. Ascites and pleural effusion samples from cancer patientsmay be tested for expression of Her2/neu, using methods known to oneskilled in the art. Samples from ovarian carcinoma patients may beprocessed by spinning down the ascites at 6370 g for 20 minutes at 4°C., lysing the red blood cells, and washing the cells with PBS. Once theexpression of Her2/neu in tumor cells is determined, two samples, amedian and a high expressor may be selected for s.c. inoculation toestablish the xenograft tumor model. The isolated tumor cells will thenbe injected i.p. into mice to expand the cells. Approximately 10 micemay be injected i.p. and each mouse ascites further passaged into twomice to obtain ascites from a total of 20 mice which can be used toinject a group of 80 mice. Pleural effusion samples may be processedusing a similar method as ascites. The Her2/neu+tumor cells from pleuraleffusion samples may be injected into the upper right and left mammarypads of the mice.

In some embodiments, if the percentage of neoplastic cells in theascites or pleural effusion samples is low compared to other cellularsubsets, the neoplastic cells may be expanded in vitro. In otherembodiments, tumor cells may be purified using CC49 antibody(anti-TAG-72)-coated magnetic beads as described previously, see, e.g.,Barker et al., 2001, Gynecol. Oncol. 82:57, 63, which is incorporatedherein by reference in its entirety. Briefly, magnetic beads coated withCC49 antibody can be used to separate the ovarian tumor cells that willbe detached from the beads by an overnight incubation at 37° C. In someembodiments, if the tumor cells lack the TAG-72 antigen, negativedepletion using a cocktail of antibodies, such as those provided by StemCell Technologies, Inc., Canada, may be used to enrich the tumor cells.

In other embodiments, other tumors markers besides Her2/neu may be usedto separate tumor cells obtained from the ascites and pleural effusionsamples from non-tumor cells. In the case of pleural effusion or breasttissue, it has been recently reported that CD44 (an adhesion molecule),B38.1(a breast/ovarian cancer-specific marker), CD24 (an adhesionmolecule) may be used as markers, see, e.g., Al Hajj, et al., 2003,Proc. Natl. Acad. Sci. USA 100:3983, 8; which is incorporated herein byreference in its entirety. Once tumor cells are purified they may beinjected s.c. into mice for expansion.

Preferably, immunohistochemistry and histochemistry is performed onascites and pleural effusion of patients to analyze structuralcharacteristics of the neoplasia. Such methods are known to one skilledin the art and encompassed within the invention. The markers that may bemonitored include for example cytokeratin (to identify ovarianneoplastic and mesothelial cells from inflammatory and mesenchymalcells); calretinin (to separate mesothelial from Her2neu positiveneoplastic cells); and CD45 (to separate inflammatory cells from therest of the cell population in the samples). Additional markers that maybe followed include CD3 (T cells), CD20 (B cells), CD56 (NK cells), andCD14 (monocytes). It will be appreciated by one skilled in the art thatthe immunohistochemistry and histochemistry methods described supra, areanalogously applied to any tumor cell for use in the methods of theinvention. After s.c. inoculation of tumor cells, mice are followed forclinical and anatomical changes. As needed, mice may be necropsied tocorrelate total tumor burden with specific organ localization.

In a specific embodiment, tumors are established using carcinoma celllines such as IGROV-1, OVCAR-8, SK-B, and OVCAR-3 cells and humanovarian carcinoma ascites and pleural effusion from breast cancerpatients. The ascites preferably contain both the effectors and thetumor targets for the antibodies being tested. Human monocytes will betransferred as effectors.

The in vivo activity of the humanized antibodies of the invention mayalso be tested in an animal model, e.g., Balb/c nude mice, injected withcells expressing FcγRIIB, including but not limited to SK-BR-3 with ATCCaccession number HTB-30 (see, e.g., Tremp et al., 1976, Cancer Res.33-41); B-lymphocytes; cells derived from Burkitts lymphoma, e.g., Rajicells with ATCC accession number CCL-86 (see, e.g., Epstein et al.,1965, J. Natl. Cancer Inst. 34: 231-240), Daudi cells with ATCCaccession number CCL-213 (see, e.g., Klein et al., 1968, Cancer Res. 28:1300-10); ovarian carcinoma cell lines, e.g., OVCAR-3 with ATCCaccession number HTB-161 (see, e.g., Hamilton, Young et al., 1983),SK-OV-3, PA-1, CAOV3, OV-90, and IGROV-1 (available from the NCIrepository Benard et al., 1985, Cancer Research, 45:4970-9; each ofwhich is incorporated herein by reference in its entirety.

An exemplary assay for measuring the in vivo activity of the humanizedantibodies of the invention may comprise the following: Balb/c Nudefemale mice (Taconic, Md.) are injected at day 0 with cells expressingFcγRIIB such as 5×10⁶ Daudi cells for example by the subcutaneous route.Mice (e.g., 5 mice per group) then receive i.p. injection of a humanizedanti-FcγRIIB antibody of the invention (e.g. a h2B6 of the invention,e.g., at 10 mg/g), i.p. injection of PBS (negative control), ch 4.4.20(anti-FITC antibody) as a negative control, and as a positive controlanother therapeutic cancer antibody such as those disclosed herein,e.g., Rituxan, (e.g., at 10 μg/g) once a week starting at day 0. Miceare observed, e.g., twice a week following injection, and tumor size(length and width) is determined using for example a caliper. Tumorweight in mg is estimated using the formula: (length×width²)/2.

Preferably, the humanized antibodies of the invention have an enhancedefficacy in decreasing tumor relative to a cancer therapeutic antibodywhen administered at the same dose, e.g., 10 μg/g, over a time period ofat least 14 days, at least 21 days, at least 28 days, or at least 35days. In most preferred embodiments, the humanized antibodies of theinvention reduce tumor size by at least 10 fold, at least 100 fold, atleast 1000 fold relative to administration of a cancer therapeuticantibody at the same dose. In yet another preferred embodiment, theantibodies of the invention completely abolish the tumor.

6.2.2 Polynucleotides Encoding an Antibody

The present invention also includes polynucleotides that encode thehumanized antibodies of the invention (e.g., mouse monoclonal antibodyproduced from clone 2B6 or 3H7, with ATCC accession numbers PTA-4591 andPTA-4592, respectively). The present invention encompass thepolynucleotide encoding the heavy chain of the 2B6 antibody, with ATCCaccession number PTA-4591 and/or the polynucleotide encoding the heavychain having the amino acid sequence SEQ ID NO:70. As a specific examplein accordance with this embodiment, the invention encompasses thepolynucleotide sequence SEQ ID NO:69, which encodes the amino acidsequende SEQ ID NO:70. The present invention also encompasses thepolynucleotide encoding the light chain of the 2B6 antibody with ATCCaccession number PTA-4591 and/or the polynucleotide encoding the lightchain having the amino acid sequence SEQ ID NO:66. As a specific examplein accordance with this embodiment, the invention encompasses thepolynucleotide sequence SEQ ID NO:65, which encodes the amino acidsequende SEQ ID NO:66. In a specific embodiment, the inventionencompasses the plasmid pMGx0675 that comprises nucleotide sequences SEQID NO:69 and SEQ ID NO:65 that encode the amino acid sequences of theheavy chain (SEQ ID NO:70) and light chain (SEQ ID NO:66), respectively,of a specific example of a h2B6 antibody of the invention, which plasmidhas ATCC Accession number PTA-7609, and was deposited on May 23, 2006.

The methods of the invention also encompass polynucleotides thathybridize under various stringency, e.g., high stringency, intermediateor lower stringency conditions, to polynucleotides that encode ahumanized antibody of the invention. The hybridization can be performedunder various conditions of stringency. By way of example and notlimitation, procedures using conditions of low stringency are as follows(see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78,6789-6792; incorporated by reference in its entirety). Filterscontaining DNA are pretreated for 6 h at 40° C. in a solution containing35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1%Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizationsare carried out in the same solution with the following modifications:0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10%(wt/vol) dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe is used.Filters are incubated in hybridization mixture for 18-20 h at 40° C.,and then washed for 1.5 h at 55° C. in a solution containing 2×SSC, 25mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 h at 60° C.Filters are blotted dry and exposed for autoradiography. If necessary,filters are washed for a third time at 65-68° C. and re-exposed to film.Other conditions of low stringency which may be used are well known inthe art (e.g., as employed for cross-species hybridizations). By way ofexample and not limitation, procedures using conditions of highstringency are as follows. Prehybridization of filters containing DNA iscarried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC,50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA,and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48h at 65° C. in prehybridization mixture containing 100 μg/ml denaturedsalmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Washing offilters is done at 37° C. for 1 h in a solution containing 2×SSC, 0.01%PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSCat 50° C. for 45 min before autoradiography. Other conditions of highstringency which may be used are well known in the art. Selection ofappropriate conditions for such stringencies is well known in the art(see e.g., Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; see also, Ausubel et al., eds., in the Current Protocols inMolecular Biology series of laboratory technique manuals, ©1987-1997,Current Protocols, ©1994-1997 John Wiley and Sons, Inc.; see especially,Dyson, 1991, “Immobilization of nucleic acids and hybridizationanalysis,” In: Essential Molecular Biology: A Practical Approach, Vol.2, T. A. Brown, ed., pp. 111-156, IRL Press at Oxford University Press,Oxford, UK (each of which is incorporated by reference in itsentirety)).

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art.

A polynucleotide encoding a humanized antibody of the invention may begenerated from nucleic acid from a suitable source (e.g., a cDNA librarygenerated from, or nucleic acid, preferably poly A+ RNA, isolated from,any tissue or cells expressing the antibody, such as hybridoma cellsselected to express an antibody of the invention, e.g., 2B6 or 3H7) byhybridization with Ig specific probes and/or PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence of the humanized antibody is determined,the nucleotide sequence of the humanized antibody may be manipulatedusing methods well known in the art for the manipulation of nucleotidesequences, e.g., recombinant DNA techniques, site directed mutagenesis,PCR, etc. (see, for example, the techniques described in Sambrook etal., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds.,1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY,which are both incorporated by reference herein in their entireties), togenerate antibodies having a different amino acid sequence, for exampleto create amino acid substitutions, deletions, and/or insertions.

In another embodiment, human libraries or any other libraries availablein the art, can be screened by standard techniques known in the art, toclone the nucleic acids encoding the antibodies of the invention.

6.2.3 Recombinant Expression of Antibodies

Once a nucleic acid sequence encoding a humanized antibody of theinvention has been obtained, the vector for the production of theantibody may be produced by recombinant DNA technology using techniqueswell known in the art, e.g. the vector pMGx0675, which encodes the heavyand light chain of a h2B6 antibody of the invention, deposited with ATCCon May 23, 2006 and having Accession number PTA-7609. Methods which arewell known to those skilled in the art can be used to constructexpression vectors containing the humanized antibody coding sequencesand appropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. (See, forexample, the techniques described in Sambrook et al., 1990, MolecularCloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. and Ausubel et al. eds., 1998, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (each of which isincorporated by reference in its entirety)).

An expression vector comprising the nucleotide sequence of a humanizedantibody of the invention can be transferred to a host cell byconventional techniques (e.g., electroporation, liposomal transfection,and calcium phosphate precipitation) and the transfected cells are thencultured by conventional techniques to produce the antibody of theinvention. In specific embodiments, the expression of the humanizedantibody is regulated by a constitutive, an inducible or a tissue,specific promoter.

The host cells used to express the recombinant antibodies of theinvention may be either bacterial cells such as Escherichia coli, or,preferably, eukaryotic cells, especially for the expression of wholerecombinant immunoglobulin molecule. In particular, mammalian cells suchas Chinese hamster ovary cells (CHO), in conjunction with a vector suchas the major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for immunoglobulins(Foecking et al., 1998, Gene 45:101; Cockett et al., 1990,Bio/Technology 8:2) (see paragraph 147). Preferably, the host cell is adihydrofolate reductase deficient chinese hamster ovary cell (CHO),e.g., a Lec 13 CHO cell (lectin resistant CHO mutant cell line; (see,e.g., U.S. Patent Application Publication No. 2003/0115614; PCTPublication No. WO 00/61739; European Patent Application EP 1 229 125;Ribka & Stanley, 1986, Somatic Cell & Molec. Gen. 12(1): 51-62; Ripka etal., 1986 Arch. Biochem. Biophys. 249(2): 533-45); each of which isincorporated by reference herein in its entirety), or a CHO-K1 cell, aDUX-B11 cell, a CHO-DP12 cell or a CHO-DG44 cell, which has beenmodified so that the antibody is not substantially fucosylated.

A variety of host-expression vector systems may be utilized to expressthe humanized antibodies of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of the humanizedantibodies may be produced and subsequently purified, but also representcells which may, when transformed or transfected with the appropriatenucleotide coding sequences, express the humanized antibodies of theinvention in situ. These include, but are not limited to, microorganismssuch as bacteria (e.g., E. coli and B. subtilis) transformed withrecombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing immunoglobulin coding sequences; yeast (e.g.,Saccharomyces Pichia) transformed with recombinant yeast expressionvectors containing immunoglobulin coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing the immunoglobulin coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingimmunoglobulin coding sequences; or mammalian cell systems (e.g., COS,CHO, BHK, 293, 293T, 3T3 cells, lymphotic cells (see U.S. Pat. No.5,807,715), Per C.6 cells (rat retinal cells developed by Crucell))harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the immunoglobulin molecule in infected hosts. (e.g., seeLogan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359; incorporatedby reference in its entirety). Specific initiation signals may also berequired for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see Bittner et al., 1987, Methods in Enzymol.153:51-544; incorporated by reference in its entirety).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 andHs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably express ahumanized antibody of the invention may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express theantibodies of the invention. Such engineered cell lines may beparticularly useful in screening and evaluation of compounds thatinteract directly or indirectly with the antibodies of the invention.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk−, hgprt− or aprt− cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, 1991, 3:87-95; Tolstoshev,1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.62:191-217; May, 1993, TIB TECH 11(5):155-215). Methods commonly knownin the art of recombinant DNA technology which can be used are describedin Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology,John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, ALaboratory Manual, Stockton Press, NY; and in Chapters 12 and 13,Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, JohnWiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1;and hygro, which confers resistance to hygromycin (Santerre et al.,1984, Gene 30:147). Each of the foregoing references is incorporatedherein by reference in its entirety.

The expression levels of a humanized antibody of the invention can beincreased by vector amplification (for a review, see Bebbington andHentschel, The use of vectors based on gene amplification for theexpression of cloned genes in mammalian cells in DNA cloning, Vol. 3.(Academic Press, New York, 1987); incorporated by reference in itsentirety). When a marker in the vector system expressing an antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the nucleotide sequence of theantibody, production of the antibody will also increase (Crouse et al.,1983, Mol. Cell. Biol. 3:257; incorporated by reference in itsentirety).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides, e.g. pMGx0675 encoding the heavy and light chain of a h2B6antibody of the invention, having ATCC accession number PTA-7609,deposited May 23, 2006. In such situations, the light chain should beplaced before the heavy chain to avoid an excess of toxic free heavychain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad.Sci. USA 77:2197; incorporated by reference in its entirety). The codingsequences for the heavy and light chains may comprise cDNA or genomicDNA.

Once the humanized antibody of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an antibody, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins.

6.3 Prophylactic and Therapeutic Methods

The present invention encompasses antibody-based therapies which involveadministering one or more of the humanized antibodies of the inventionto an animal, preferably a mammal, and most preferably a human, forpreventing, treating, or ameliorating symptoms associated with adisease, disorder, or infection, associated with aberrant levels oractivity of FcγRIIB and/or treatable by altering immune functionassociated with FcγRIIB activity or enhancing cytotoxic activity of asecond therapeutic antibody or enhancing efficacy of a vaccinecomposition or breaking tolerance to an antigen. In some embodiments,therapy by administration of one or more antibodies of the invention iscombined with administration of one or more therapies such as, but notlimited to, chemotherapies, radiation therapies, hormonal therapies,and/or biological therapies/immunotherapies.

FcγRIIB (CD32B) has been found to be expressed in the following tissuetypes: adipose, b-cell, bone, brain, cartilage, colon, endocrine, eye,fetus, gastrointestinal tract, genitourinary, germ cell, head and neck,kidney, lung, lymph node, lymphoreticular, mammary gland, muscle,nervous, ovary, pancreas, pancreatic islet, pituitary gland, placenta,retina, skin, soft tissue, synovium, and uterus (data collected from theCancer Genome Anatomy Project of the National Cancer Institute). Thus,the humanized antibodies of the invention can be used to agonize orantagonize the activity of FcγRIIB in any of these tissues. For example,FcγRIIB is expressed in the placenta and may play a role in transport ofIgG to the fetus and also in scavenging immune complexes (Lyden et al.,2001, J. Immunol. 166:3882-3889). In certain embodiments of theinvention, a humanized FcγRIIB antibody can used as an abortifacient.

Prophylactic and therapeutic compounds of the invention include, but arenot limited to, proteinaceous molecules, including, but not limited to,peptides, polypeptides, proteins, including post-translationallymodified proteins, antibodies, etc.; small molecules (less than 1000daltons), inorganic or organic compounds; nucleic acid moleculesincluding, but not limited to, double-stranded or single-stranded DNA,double-stranded or single-stranded RNA, as well as triple helix nucleicacid molecules. Prophylactic and therapeutic compounds can be derivedfrom any known organism (including, but not limited to, animals, plants,bacteria, fungi, and protista, or viruses) or from a library ofsynthetic molecules.

Humanized antibodies may be provided in pharmaceutically acceptablecompositions as known in the art or as described herein. As detailedbelow, the humanized antibodies of the invention can be used in methodsof treating cancer (particularly to enhance passive immunotherapy orefficacy of a cancer vaccine), autoimmune disease, inflammatorydisorders or allergies (e.g., to enhance efficacy of a vaccine fortreatment of allergy).

Humanized antibodies of the present invention that function as aprophylactic and/or therapeutic agent of a disease, disorder, orinfection can be administered to an animal, preferably, a mammal andmost preferably, a human, to treat, prevent or ameliorate one or moresymptoms associated with the disease, disorder, or infection. Antibodiesof the invention can be administered in combination with one or moreother prophylactic and/or therapeutic agents useful in the treatment,prevention or management of a disease, disorder, or infection associatedwith aberrant levels or activity of FcγRIIB and/or treatable by alteringimmune function associated with FcγRIIB activity. In certainembodiments, one or more antibodies of the invention are administered toa mammal, preferably, a human, concurrently with one or more othertherapeutic agents useful for the treatment of cancer. The term“concurrently” is not limited to the administration of prophylactic ortherapeutic agents at exactly the same time, but rather it is meant thatantibodies of the invention and the other agent are administered to asubject in a sequence and within a time interval such that theantibodies of the invention can act together with the other agent toprovide an increased benefit than if they were administered otherwise.For example, each prophylactic or therapeutic agent may be administeredat the same time or sequentially in any order at different points intime; however, if not administered at the same time, they should beadministered sufficiently close in time so as to provide the desiredtherapeutic or prophylactic effect. Each therapeutic agent can beadministered separately, in any appropriate form and by any suitableroute.

In various embodiments, the prophylactic or therapeutic agents areadministered less than 1 hour apart, at about 1 hour apart, at about 1hour to about 2 hours apart, at about 2 hours to about 3 hours apart, atabout 3 hours to about 4 hours apart, at about 4 hours to about 5 hoursapart, at about 5 hours to about 6 hours apart, at about 6 hours toabout 7 hours apart, at about 7 hours to about 8 hours apart, at about 8hours to about 9 hours apart, at about 9 hours to about 10 hours apart,at about 10 hours to about 11 hours apart, at about 11 hours to about 12hours apart, no more than 24 hours apart or no more than 48 hours apart.In preferred embodiments, two or more components are administered withinthe same patient visit.

The dosage amounts and frequencies of administration provided herein areencompassed by the terms therapeutically effective and prophylacticallyeffective. The dosage and frequency further will typically varyaccording to factors specific for each patient depending on the specifictherapeutic or prophylactic agents administered, the severity and typeof cancer, the route of administration, as well as age, body weight,response, and the past medical history of the patient. Suitable regimenscan be selected by one skilled in the art by considering such factorsand by following, for example, dosages reported in the literature andrecommended in the Physician's Desk Reference (56^(th) ed., 2002).

The humanized antibodies of this invention may also be advantageouslyutilized in combination with other monoclonal or chimeric antibodies, Fcfusion proteins, or with lymphokines, cytokines or hematopoietic growthfactors (such as, e.g., IL-2, IL-3, IL-4, IL-7, IL-10 and TGF-β), whichenhance FcγRIIB, for example, serve to increase the number or activityof effector cells which interact with the antibodies and, increaseimmune response. In certain embodiments, a cytokine is conjugated to ananti-FcγRIIB antibody.

The humanized antibodies of this invention may also be advantageouslyutilized in combination with one or more drugs used to treat a disease,disorder, or infection such as, for example anti-cancer agents,anti-inflammatory agents or anti-viral agents, e.g., as detailed insections 5.3.6 and 5.3.5 below.

6.3.1 Cancers

Humanized antibodies of the invention can be used alone or incombination with other therapeutic antibodies known in the art toprevent, inhibit or reduce the growth of primary tumores or metastasisof cancerous cells. In one embodiment, humanized antibodies of theinvention can be used in combination with antibodies used in cancerimmunotherapy. The invention encompasses the use of the humanizedantibodies of the invention in combination with another therapeuticantibody to enhance the efficacy of such immunotherapy by increasing thepotency of the therapeutic antibody's effector function, e.g., ADCC,CDC, phagocytosis, opsonization, etc. Although not intending to be boundby a particular mechanism of action antibodies of the invention blockFcγRIIB, preferably on monocytes and macrophages and thus enhance thetherapeutic benefits a clinical efficacy of tumor specific antibodiesby, for example, enhancing clearance of the tumors mediated byactivating fcγRs.

Accordingly, the invention provides methods of preventing or treatingcancer characterized by a cancer antigen, when administered incombination with another antibody that specifically binds a cancerantigen and is cytotoxic. The humanized antibodies of the invention areuseful for prevention or treatment of cancer, particularly inpotentiating the cytotoxic activity of cancer antigen-specifictherapeutic antibodies with cytotoxic activity to enhance tumor cellkilling by the antibodies of the invention and/or enhancing, forexample, ADCC activity or CDC activity of the therapeutic antibodies. Incertain embodiments of the invention, humanized antibodies of theinvention are administered with Fc fusion proteins. In a specificembodiment, a humanized antibody of the invention, when administeredalone or in combination with a cytotoxic therapeutic antibody, inhibitsor reduces the growth of primary tumor or metastasis of cancerous cellsby at least 99%, at least 95%, at least 90%, at least 85%, at least 80%,at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, atleast 40%, at least 45%, at least 35%, at least 30%, at least 25%, atleast 20%, or at least 10% relative to the growth of primary tumor ormetastasis in absence of said antibody of the invention. In a preferredembodiment, humanized antibodies of the invention in combination with acytotoxic therapeutic antibody inhibit or reduce the growth of primarytumor or metastasis of cancer by at least 99%, at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, at least 50%, at least 45%, at least 40%, at least 45%, at least35%, at least 30%, at least 25%, at least 20%, or at least 10% relativeto the growth or metastasis in absence of said antibodies.

The transition from a normal to a malignant state is a multistep processinvolving genetic and epigenetic changes. In fact, numerous alterationsoccur in the cellular regulatory circuits that facilitate thisprogression which enables tumor cells to evade the commitment toterminal differentiation and quiescence that normally regulate tissuehomeostasis. Certain genes have been implicated in invasiveness andmetastatic potential of cancer cells such as CSF-1 (colony stimulatingfactor 1 or macrophage colony stimulating factor). Although notintending to be bound by a particular mechanism of action, CSF-1 maymediate tumor progression and metastasis by recruiting macrophages tothe tumor site where they promote progression of tumor. It is believedthat macrophages have a trophic role in mediating tumor progression andmetastasis perhaps by the secretion of angiogenic factors, e.g.,thymidine phosphorylase, vascular endothelial-derived growth factor;secretion of growth factors such as epidermal growth factor that couldact as a paracrine factor on tumor cells, and thus promoting tumor cellmigration and invasion into blood vessels. (See, e.g., Lin et al., 2001,J. Exp. Med. 193(6): 727-739; Lin et al., 2002, Journal of Mammary GlandBiology and Neoplasam 7(2): 147-162; Scholl et al., 1993, MolecularCarcinogenesis, 7: 207-11; Clynes et al., 2000, Nature Medicine, 6(4):443-446; Fidler et al., 1985, Cancer Research, 45: 4714-26 (each ofwhich is incorporated by reference in its entirety)).

The invention encompasses using the humanized antibodies of theinvention to block macrophage mediated tumor cell progression andmetastasis. The antibodies of the invention are particularly useful inthe treatment of solid tumors, where macrophage infiltration occurs. Theantagonistic antibodies of the invention are particularly useful forcontrolling, e.g., reducing or eliminating, tumor cell metastasis, byreducing or eliminating the population of macrophages that are localizedat the tumor site. In some embodiments, the humanized antibodies of theinvention are used alone to control tumor cell metastasis. Although notintending to be bound by a particular mechanism of action, theantagonistic antibodies of the invention, when administered alone bindthe inhibitory FcγRIIB on macrophages and effectively reduce thepopulation of macrophages and thus restrict tumor cell progression. Theantagonistic antibodies of the invention reduce, or preferably,eliminate macrophages that are localized at the tumor site, sinceFcγRIIB is preferentially expressed on activated monocytes andmacrophages including tumor-infiltrating macrophages. In someembodiments, the humanized antibodies of the invention are used in thetreatment of cancers that are characterized by the overexpression ofCSF-1, including, but not limited to, breast, uterine, and ovariancancers.

The invention further encompasses humanized antibodies that effectivelydeplete or eliminate immune cells other than macrophages that expressFcγRIIB, e.g., dendritic cells and B-cells. Effective depletion orelimination of immune cells using the antibodies of the invention mayrange from a reduction in population of the immune cells by 50%, 60%,70%, 80%, preferably 90%, and most preferably 99%. Thus, the humanizedantibodies of the invention have enhanced therapeutic efficacy eitheralone or in combination with a second antibody, e.g., a therapeuticantibody such as anti-tumor antibodies, anti-viral antibodies, andanti-microbial antibodies. In some embodiments, the therapeuticantibodies have specificity for a cancer cell or an inflammatory cell.In other embodiments, the second antibody binds a normal cell. Althoughnot intending to be bound by a particular mechanism of action, when theantibodies of the invention are used alone to deplete FcγRIIB-expressingimmune cells, the population of cells is redistributed so thateffectively the cells that are remaining have the activating Fcreceptors and thus the suppression by FcγRIIB is alleviated. When usedin combination with a second antibody, e.g., a therapeutic antibody theefficacy of the second antibody is enhanced by increasing theFc-mediated effector function of the antibody.

The humanized antibodies and fragments thereof of the invention andmethods of treatment ae believed to be effective for the treatment ofboth liquid and solid cancers. By liquid cancers it is meant cancers ofthe bone marrow, such as leukemias. Solid cancers generally refer tocancers of organs and/or other tissues. Cancers and related disordersthat can be treated or prevented by methods and compositions of thepresent invention include, but are not limited to, the following:leukemias including, but not limited to, acute leukemia, acutelymphocytic leukemia, acute myelocytic leukemias such as myeloblastic,promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias andmyelodysplastic syndrome, chronic leukemias such as but not limited to,chronic myelocytic (granulocytic) leukemia, chronic lymphocyticleukemia, hairy cell leukemia; polycythemia vera; lymphomas such as, butnot limited to, Hodgkin's disease, non-Hodgkin's disease; multiplemyelomas such as, but not limited to, smoldering multiple myeloma,nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia,solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom'smacroglobulinemia; monoclonal gammopathy of undetermined significance;benign monoclonal gammopathy; heavy chain disease; bone and connectivetissue sarcomas such as, but not limited to, bone sarcoma, osteosarcoma,chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor,fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissuesarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi'ssarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma,rhabdomyosarcoma, synovial sarcoma; brain tumors including but notlimited to, glioma, astrocytoma, brain stem glioma, ependymoma,oligodendroglioma, nonglial tumor, acoustic neurinoma,craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including, but notlimited to, adenocarcinoma, lobular (small cell) carcinoma, intraductalcarcinoma, medullary breast cancer, mucinous breast cancer, tubularbreast cancer, papillary breast cancer, Paget's disease, andinflammatory breast cancer; adrenal cancer, including but not limitedto, pheochromocytom and adrenocortical carcinoma; thyroid cancer such asbut not limited to papillary or follicular thyroid cancer, medullarythyroid cancer and anaplastic thyroid cancer; pancreatic cancer,including but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers including but not limited to, Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers including, but not limited to, ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers, including, but not limited to, squamouscell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, includingbut not limited to, squamous cell carcinoma, melanoma, adenocarcinoma,basal cell carcinoma, sarcoma, and Paget's disease; cervical cancersincluding, but not limited to, squamous cell carcinoma, andadenocarcinoma; uterine cancers including, but not limited to,endometrial carcinoma and uterine sarcoma; ovarian cancers including,but not limited to, ovarian epithelial carcinoma, borderline tumor, germcell tumor, and stromal tumor; esophageal cancers including, but notlimited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma,mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma,plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma;stomach cancers including, but not limited to, adenocarcinoma, fungating(polypoid), ulcerating, superficial spreading, diffusely spreading,malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; coloncancers; rectal cancers; liver cancers including, but not limited to,hepatocellular carcinoma and hepatoblastoma, gallbladder cancersincluding, but not limited to, adenocarcinoma; cholangiocarcinomasincluding, but not limited to, pappillary, nodular, and diffuse; lungcancers including but not limited to, non-small cell lung cancer,squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma,large-cell carcinoma and small-cell lung cancer; testicular cancersincluding, but not limited to, germinal tumor, seminoma, anaplastic,classic (typical), spermatocytic, nonseminoma, embryonal carcinoma,teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancersincluding, but not limited to, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers including, but not limitedto, squamous cell carcinoma; basal cancers; salivary gland cancersincluding, but not limited to, adenocarcinoma, mucoepidermoid carcinoma,and adenoidcystic carcinoma; pharynx cancers including, but not limitedto, squamous cell cancer, and verrucous; skin cancers including, but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers including, but notlimited to, renal cell cancer, adenocarcinoma, hypernephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor; bladder cancers including, but not limited to,transitional cell carcinoma, squamous cell cancer, adenocarcinoma,carcinosarcoma. In addition, cancers include myxosarcoma, osteogenicsarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma,synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma,bronchogenic carcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma and papillary adenocarcinomas (for areview of such disorders, see Fishman et al., 1985, Medicine, 2d Ed.,J.B. Lippincott Co., Philadelphia and Murphy et al., 1997, InformedDecisions: The Complete Book of Cancer Diagnosis, Treatment, andRecovery, Viking Penguin, Penguin Books U.S.A., Inc., United States ofAmerica (each of which is incorporated by reference in its entirety)).

Accordingly, the methods and compositions of the invention are alsouseful in the treatment or prevention of a variety of cancers or otherabnormal proliferative diseases, including (but not limited to) thefollowing: carcinoma, including that of the bladder, breast, colon,kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin;including squamous cell carcinoma; hematopoietic tumors of lymphoidlineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Berkettslymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including melanoma, seminoma, tetratocarcinoma, neuroblastomaand glioma; tumors of the central and peripheral nervous system,including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin, including fibrosafcoma, rhabdomyoscarama, andosteosarcoma; and other tumors, including melanoma, xenodermapegmentosum, keratoactanthoma, seminoma, thyroid follicular cancer andteratocarcinoma. It is also contemplated that cancers caused byaberrations in apoptosis would also be treated by the methods andcompositions of the invention. Such cancers may include, but are not belimited to, follicular lymphomas, carcinomas with p53 mutations, hormonedependent tumors of the breast, prostate and ovary, and precancerouslesions such as familial adenomatous polyposis, and myelodysplasticsyndromes. In specific embodiments, malignancy or dysproliferativechanges (such as metaplasias and dysplasias), or hyperproliferativedisorders, are treated or prevented by the methods and compositions ofthe invention in the ovary, bladder, breast, colon, lung, skin,pancreas, or uterus. In other specific embodiments, sarcoma, melanoma,or leukemia is treated or prevented by the methods and compositions ofthe invention.

Cancers associated with the cancer antigens may be treated or preventedby administration of the antibodies of the invention in combination withan antibody that binds the cancer antigen and is cytotoxic. In oneparticular embodiment, the antibodies of the invention enhance theantibody mediated cytotoxic effect of the antibody directed at theparticular cancer antigen. For example, but not by way of limitation,cancers associated with the following cancer antigens may be treated orprevented by the methods and compositions of the invention: KS 1/4pan-carcinoma antigen (Perez and Walker, 1990, J. Immunol. 142:32-37;Bumal, 1988, Hybridoma 7(4):407-415), ovarian carcinoma antigen (CA125)(Yu et al., 1991, Cancer Res. 51(2):48-475), prostatic acid phosphate(Tailor et al., 1990, Nucl. Acids Res. 18(1):4928), prostate specificantigen (Henttu and Vihko, 1989, Biochem. Biophys. Res. Comm.10(2):903-910; Israeli et al., 1993, Cancer Res. 53:227-230),melanoma-associated antigen p97 (Estin et al., 1989, J. Natl. CancerInstit. 81(6):445-44), melanoma antigen gp75 (Vijayasardahl et al.,1990, J. Exp. Med. 171(4):1375-1380), high molecular weight melanomaantigen (HMW-MAA) (Natali et al., 1987, Cancer 59:55-3; Mittelman etal., 1990, J. Clin. Invest. 86:2136-2144)), prostate specific membraneantigen, carcinoembryonic antigen (CEA) (Foon et al., 1994, Proc. Am.Soc. Clin. Oncol. 13:294), polymorphic epithelial mucin antigen, humanmilk fat globule antigen, Colorectal tumor-associated antigens such as:CEA, TAG-72 (Yokata et al., 1992, Cancer Res. 52:3402-3408), C017-1A(Ragnhammar et al., 1993, Int. J. Cancer 53:751-758); GICA 19-9 (Herlynet al., 1982, J. Clin. Immunol. 2:135), CTA-1 and LEA, Burkitt'slymphoma antigen-38.13, CD19 (Ghetie et al., 1994, Blood 83:1329-1336),human B-lymphoma antigen-CD20 (Reff et al., 1994, Blood 83:435-445),CD33 (Sgouros et al., 1993, J. Nucl. Med. 34:422-430), melanoma specificantigens such as ganglioside GD2 (Saleh et al., 1993, J. Immunol., 151,3390-3398), ganglioside GD3 (Shitara et al., 1993, Cancer Immunol.Immunother. 36:373-380), ganglioside GM2 (Livingston et al., 1994, J.Clin. Oncol. 12:1036-1044), ganglioside GM3 (Hoon et al., 1993, CancerRes. 53:5244-5250), tumor-specific transplantation type of cell-surfaceantigen (TSTA) such as virally-induced tumor antigens includingT-antigen DNA tumor viruses and envelope antigens of RNA tumor viruses,oncofetal antigen-alpha-fetoprotein such as CEA of colon, bladder tumoroncofetal antigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188),differentiation antigen such as human lung carcinoma antigen L6, L20(Hellstrom et al., 1986, Cancer Res. 46:3917-3923), antigens offibrosarcoma, human leukemia T cell antigen-Gp37(Bhattacharya-Chatterjee et al., 1988, J. of Immun. 141:1398-1403),neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR(Epidermal growth factor receptor), HER2 antigen (p185^(HER2)),polymorphic epithelial mucin (PEM) (Hilkens et al., 1992, Trends in Bio.Chem. Sci. 17:359), malignant human lymphocyte antigen-APO-1 (Bernhardet al., 1989, Science 245:301-304), differentiation antigen (Feizi,1985, Nature 314:53-57) such as I antigen found in fetal erthrocytes andprimary endoderm, I(Ma) found in gastric adencarcinomas, M18 and M39found in breast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9,Myl, VIM-D5, and D₁56-22 found in colorectal cancer, TRA-1-85 (bloodgroup H), C14 found in colonic adenocarcinoma, F3 found in lungadenocarcinoma, AH6 found in gastric cancer, Y hapten, Le^(y) found inembryonal carcinoma cells, TL5 (blood group A), EGF receptor found inA431 cells, E₁ series (blood group B) found in pancreatic cancer, FC10.2found in embryonal carcinoma cells, gastric adenocarcinoma, CO-514(blood group Le^(a)) found in adenocarcinoma, NS-10 found inadenocarcinomas, CO-43 (blood group Le^(b)), G49, EGF receptor, (bloodgroup ALe^(b)/Le^(y)) found in colonic adenocarcinoma, 19.9 found incolon cancer, gastric cancer mucins, T₅A₇ found in myeloid cells, R₂₄found in melanoma, 4.2, G_(D3), D1.1, OFA-1, G_(M2), OFA-2, G_(D2),M1:22:25:8 found in embryonal carcinoma cells and SSEA-3, SSEA-4 foundin 4-8-cell stage embryos. In another embodiment, the antigen is a Tcell receptor derived peptide from a cutaneous T cell lymphoma (seeEdelson, 1998, The Cancer Journal 4:62). Each of the foregoingreferences is hereby incorporated herein in its entirety.

The humanized antibodies of the invention can be used in combinationwith any therapeutic cancer antibodies known in the art to enhance theefficacy of treatment. For example, the humanized antibodies of theinvention can be used with any of the antibodies in Table 4 that havedemonstrated therapeutic utility in cancer treatment. The humanizedantibodies of the invention enhance the efficacy of treatment of thetherapeutic cancer antibodies by enhancing at least oneantibody-mediated effector function of said therapeutic cancerantibodies. In one particular embodiment, the humanized antibodiesenhance the efficacy of treatment by enhancing the complement dependentcascade of said therapeutic cancer antibodies. In another embodiment ofthe invention, the humanized antibodies of the invention enhance theefficacy of treatment by enhancing the phagocytosis and opsonization ofthe targeted tumor cells. In another embodiment of the invention, thehumanized antibodies of the invention enhance the efficacy of treatmentby enhancing antibody-dependent cell-mediated cytotoxicity (“ADCC”) indestruction of the targeted tumor cells.

Humanized antibodies of the invention can also be used in combinationwith cytosine-guanine dinucleotides (“CpG”)-based products that havebeen developed (Coley Pharmaceuticals) or are currently being developedas activators of innate and acquired immune responses. For example, theinvention encompasses the use of CpG 7909, CpG 8916, CpG 8954 (ColeyPharmaceuticals) in the methods and compositions of the invention forthe treatment and/or prevention of cancer (See also Warren et al., 2002,Semin Oncol., 29(1 Suppl 2):93-7; Warren et al., 2000, Clin Lymphoma,1(1):57-61, both of which are incorporated herein by reference).

Humanized antibodies of the invention can be used in combination with atherapeutic antibody that does not mediate its therapeutic effectthrough cell killing to potentiate the antibody's therapeutic activity.In a specific embodiment, the invention encompasses use of theantibodies of the invention in combination with a therapeutic apoptosisinducing antibody with agonisitc activity, e.g., an anti-Fas antibody.Anti-Fas antibodies are known in the art and include, for example, Jo2(Ogasawara et al., 1993, Nature 364: 806) and HFE7 (Ichikawa et al.,2000, Int. Immunol. 12: 555) (each of which is incorporated by referencein its entirety). Although not intending to be bound by a particularmechanisms of action, FcγRIIB has been implicated in promoting anti-Fasmediated apoptosis, see, e.g., Xu et al., 2003, Journal of Immunology,171: 562-568; incorporated by reference in its entirety. In fact, theextracellular domain of FcγRIIB may serve as a cross-linking agent forFas receptors, leading to a functional complex and promoting Fasdependent apoptosis. In some embodiments, the antibodies of theinvention block the interaction of anti-Fas antibodies and FcγRIIB,leading to a reduction in Fas-mediated apoptotic activity. Antibodies ofthe invention that result in a reduction in Fas-mediated apoptoticactivity are particularly useful in combination with anti-Fas antibodiesthat have undesirable side effects, e.g., hepatotoxicity. In otherembodiments, the antibodies of the invention enhance the interaction ofanti-Fas antibodies and FcγRIIB, leading to an enhancement ofFas-mediated apoptotic activity. Combination of the antibodies of theinvention with therapeutic apoptosis inducing antibodies with agonisitcactivity have an enhanced therapeutic efficacy.

Therapeutic apoptosis inducing antibodies used in the methods of theinvention may be specific for any death receptor known in the art forthe modulation of apoptotic pathway, e.g., TNFR receptor family.

The invention provides a method of treating diseases with impairedapoptotic mediated signaling, e.g., cancer or autoimmune disease. In aspecific embodiment, the invention encompasses a method of treating adisease with deficient Fas-mediated apoptosis, said method comprisingadministering an antibody of the invention in combination with ananti-Fas antibody.

In some embodiments, the agonistic antibodies of the invention areparticularly useful for the treatment of tumors of non-hematopoieticorigin, including tumors of melanoma cells. Although not intending to bebound by a particular mechanism of action, the efficacy of the agonisticantibodies of the invention is due, in part, to activation of FcγRIIBinhibitory pathway, as tumors of non-hematopoietic origin, includingtumors of melanoma cells express FcγRIIB. Recent experiments have infact shown that expression of FcγRIIB in melanoma cells modulates tumorgrowth by direct interaction with anti-tumor antibodies (e.g., bybinding the Fc region of the anti-tumor antibodies) in anintracytoplasmic-dependent manner (Cassard et al., 2002, Journal ofClinical Investigation, 110(10): 1549-1557; incorporated by reference inits entirety).

In some embodiments, the invention encompasses use of the antibodies ofthe invention in combination with therapeutic antibodies thatimmunospecifically bind to tumor antigens that are not expressed on thetumor cells themselves, but rather on the surrounding reactive and tumorsupporting non-malignant cells comprising the tumor stroma. The tumorstroma comprises endothelial cells forming new blood vessels and stromalfibroblasts surrounding the tumor vasculature. In a specific embodiment,an antibody of the invention is used in combination with an antibodythat immunospecifically binds a tumor antigen on an endothelial cell. Ina preferred embodiment, an antibody of the invention is used incombination with an antibody that immunospecifically binds a tumorantigen on a fibroblast cell, e.g., fibroblast activation protein (FAP).FAP is a 95 KDa homodimeric type II glycoprotein which is highlyexpressed in stromal fibroblasts of many solid tumors, including, butnot limited to, lung, breast, and colorectal carcinomas. (See, e.g.,Scanlan et al., 1994; Proc. Natl. Acad. USA, 91: 5657-61; Park et al.,1999, J. Biol. Chem., 274: 36505-12; Rettig et al., 1988, Proc. Natl.Acad. Sci. USA 85: 3110-3114; Garin-Chesea et al., 1990, Proc. Natl.Acad. Sci. USA 87: 7235-7239). Antibodies that immunospecifically bindFAP are known in the art and encompassed within the invention, see,e.g., Wuest et al., 2001, Journal of Biotechnology, 159-168; Mersmann etal., 2001, Int. J. Cancer, 92: 240-248; U.S. Pat. No. 6,455,677; all ofwhich are incorporated herein in by reference in their entireties.

Recently IgE's have been implicated as mediators of tumor growth and, infact, IgE-targeted immediate hypersensitivity and allergic inflammationreactions have been proposed as possible natural mechanisms involved inanti-tumor responses (for a review, see, e.g., Mills et al., 1992, Am.Journal of Epidemiol. 122: 66-74; Eriksson et al., 1995, Allergy 50:718-722 (each of which is incorporated by reference in its entirety)).In fact, a recent study has shown loading tumor cells with IgEs reducestumor growth, leading in some instances to tumor rejection. According tothe study, IgE loaded tumor cells not only possess a therapeuticpotential but also confer long term antitumor immunity, includingactivation of innate immunity effector mechanism and T-cell mediatedadaptive immune response, see Reali et al., 2001, Cancer Res. 61:5516-22; which is incorporated herein by reference in its entirety. Theantagonistic antibodies of the invention may be used in the treatmentand/or prevention of cancer in combination with administration of IgEsin order to enhance the efficacy of IgE-mediated cancer therapy.Although not intending to be bound by a particular mechanism of actionthe antibodies of the invention enhance the therapeutic efficacy of IgEtreatment of tumors, by blocking the inhibitory pathway. Theantagonistic antibodies of the invention may enhance the therapeuticefficacy of IgE mediated cancer therapy by (i) enhancing the delay intumor growth; (ii) enhancing the decrease in the rate of tumorprogression; (iii) enhancing tumor rejection; or (iv) enhancingprotective immune relative to treatment of cancer with IgE alone.

Cancer therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in theliterature, see, e.g., Physician's Desk Reference (56^(th) ed., 2002,which is incorporated herein by reference).

6.3.2 B Cell Malignancies

The agonistic antibodies of the invention are useful for treating orpreventing any B cell malignancies, particularly non-Hodgkin's lymphomaand chronic lymphocytic leukemia. Other B-cell malignancies includesmall lymphocytic lymphoma, Burkitt's lymphoma, mantle cell lymphomasdiffuse small cleaved cell lymphomas, most follicular lymphomas and somediffuse large B cell lymphomas (DLBCL). FcγRIIB, is a target forderegulation by chromosomal translocation in malignant lymphoma,particularly in B-cell non-Hodgkin's lymphoma (See Callanan M. B. etal., 2000 Proc. Natl. Acad. Sci. U.S.A., 97(1):309-314). Thus, theantibodies of the invention are useful for treating or preventing anychronic lymphocytic leukemia of the B cell lineage. Chronic lymphocyticleukemia of the B cell lineage are reviewed by Freedman (See review byFreedman, 1990, Hemtaol. Oncol. Clin. North Am. 4:405). Although notintending to be bound by any mechanism of action, the agonisticantibodies of the invention inhibit or prevent B cell malignanciesinhibiting B cell proliferation and/or activation. The invention alsoencompasses the use of the agonistic antibodies of the invention incombination with other therapies known (e.g., chemotherapy andradiotherapy) in the art for the prevention and/or treatment of B cellmalignancies. The invention also encompasses the use of the agonisticantibodies of the invention in combination with other antibodies knownin the art for the treatment and or prevention of B-cell malignancies.For example, the agonistic antibodies of the invention can be used incombination with the anti-C22 or anti-CD19 antibodies disclosed byGoldenberg et al. (U.S. Pat. No. 6,306,393), anti-CD20 antibodies,anti-CD33 antibodies, or anti-CD52 antibodies.

Antibodies of the invention can also be used in combination with, forexample, Oncoscint (target: CEA), Verluma (target: GP40), Prostascint(target: PSMA), CEA-SCAN (target: CEA), Rituxin (target: CD20),Herceptin (target: HER-2), Campath (target: CD52), Mylotarge (target:CD33), LymphoCide (target: CD22), Lymphocide Y-90 (target: CD22) andZevalin (target: CD20).

6.3.3 Autoimmune Disease and Inflammatory Diseases

The agonistic antibodies of the invention may be used to treat orprevent autoimmune diseases or inflammatory diseases. The presentinvention provides methods of preventing, treating, or managing one ormore symptoms associated with an autoimmune or inflammatory disorder ina subject, comprising administering to said subject a therapeuticallyeffective amount of the antibodies or fragments thereof of theinvention. The invention also provides methods for preventing, treating,or managing one or more symptoms associated with an inflammatorydisorder in a subject further comprising, administering to said subjecta therapeutically effective amount of one or more anti-inflammatoryagents. The invention also provides methods for preventing, treating, ormanaging one or more symptoms associated with an autoimmune diseasefurther comprising, administering to said subject a therapeuticallyeffective amount of one or more immunomodulatory agents. Section 5.4.5provides non-limiting examples of anti-inflammatory agents andimmunomodulatory agents.

The humanized antibodies of the invention can also be used incombination with any of the antibodies known in the art for thetreatment and/or prevention of autoimmune disease or inflammatorydisease. A non-limiting example of the antibodies or Fc fusion proteinsthat are used for the treatment or prevention of inflammatory disordersis presented in Table 4A, and a non-limiting example of the antibodiesor Fc fusion proteins that are used for the treatment or prevention ofautoimmune disorder is presented in Table 4B. The humanized antibodiesof the invention can, for example, enhance the efficacy of treatment ofthe therapeutic antibodies or Fc fusion proteins presented in Tables 5Aand 5B. For example, but not by way of limitation, the antibodies of theinvention can enhance the immune response in the subject being treatedwith any of the antibodies or Fc fusion proteins in Tables 5A or 5B.

Humanized antibodies of the invention can also be used in combinationwith for example, but not by way of limitation, Orthoclone OKT3, ReoPro,Zenapax, Simulec, Rituximab, Synagis, and Remicade.

Humanized antibodies of the invention can also be used in combinationwith cytosine-guanine dinucleotides (“CpG”)-based products that havebeen developed (Coley Pharmaceuticals) or are currently being developedas activators of innate and acquired immune responses. For example, theinvention encompasses the use of CpG 7909, CpG 8916, CpG 8954 (ColeyPharmaceuticals) in the methods and compositions of the invention forthe treatment and/or prevention of autoimmune or inflammatory disorders(Weeratna et al., 2001, FEMS Immunol Med. Microbial., 32(1):65-71, whichis incorporated herein by reference).

Examples of autoimmune disorders that may be treated by administeringthe antibodies of the present invention include, but are not limited to,alopecia greata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, autoimmune diseases of the adrenal gland,autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritisand orchitis, autoimmune thrombocytopenia, Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto'sthyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupuserthematosus, Meniere's disease, mixed connective tissue disease,multiple sclerosis, type 1 or immune-mediated diabetes mellitus,myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritisnodosa, polychrondritis, polyglandular syndromes, polymyalgiarheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoidarthritis (including rheumatoid arthritis of the skin, eyes, lungs,heart, blood or nerves), sarcoidosis, scleroderma, Sjögren's syndrome,stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus,takayasu arteritis, temporal arteristis/giant cell arteritis, ulcerativecolitis, uveitis, vasculitides such as dermatitis herpetiformisvasculitis, vitiligo, and Wegener's granulomatosis. Examples ofinflammatory disorders include, but are not limited to, asthma,encephilitis, inflammatory bowel disease, chronic obstructive pulmonarydisease (COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentiated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacteria infections. As described herein inSection 3.1, some autoimmune disorders are associated with aninflammatory condition. Thus, there is overlap between what isconsidered an autoimmune disorder and an inflammatory disorder.Therefore, some autoimmune disorders may also be characterized asinflammatory disorders. Examples of inflammatory disorders which can beprevented, treated or managed in accordance with the methods of theinvention include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentiated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacteria infections. The term arthritis is alsogenerally used to describe disorders beyond rheumatoid arthritis, thatare commonly associated with autoimmune inflammation. Nonlimitingexamples of such disorders that can be prevented, treated or managed inaccordance with the methods of the invention include psoriaticarthritis, Reiter's syndrome and ankylosing spondylitis arthritis.

Use of the invention in the context of an autoimmune disease (e.g.,rheumatoid arthritis) therefore excompasses methods to treat the acutephases of the disease as well as to prevent recurrence of its symptoms.The response to the therapeutic methods of the invention in the contextof the autoimmune disease (e.g., rheumatoid arthritis) may be assessedby methods described herein or by any method known to one of ordinaryskill in the art. For example, in the case of rheumatoid arthritis,many, but not all, people have rheumatoid-factor antibody in theirblood; however, the presence of rheumatoid factor is not in itselfdefinitive for a positive diagnosis of RA as other conditions are knowwhich cause the rheumatoid factor to be produced. Therefore, thediagnosis and assessment of rheumatoid arthritis is most commonly basedon a combination of factors, including, but not limited to: the specificlocation and symmetry of painful joints, the presence of joint stiffnessin the morning, the presence of bumps and nodules under the skin(rheumatoid nodules), results of X-ray tests that suggest rheumatoidarthritis.

In certain embodiments of the invention, the humanized antibodies of theinvention may be used to treat an autoimmune disease that is moreprevalent in one sex. For example, the prevalence of Graves' disease inwomen has been associated with expression of FcγRIIB2 (see Estienne etal., 2002, FASEB J. 16:1087-1092; incorporated by reference in itsentirety). Accordingly, humanized antibodies of the invention may beused to treat, prevent, ameliorate, or manage Graves' disease.

Humanized antibodies of the invention can also be used to reduce theinflammation experienced by animals, particularly mammals, withinflammatory disorders. In a specific embodiment, an antibody reducesthe inflammation in an animal by at least 99%, at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, at least 50%, at least 45%, at least 40%, at least 45%, at least35%, at least 30%, at least 25%, at least 20%, or at least 10% relativeto the inflammation in an animal in the not administered said antibody.In another embodiment, a combination of antibodies reduce theinflammation in an animal by at least 99%, at least 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, atleast 50%, at least 45%, at least 40%, at least 45%, at least 35%, atleast 30%, at least 25%, at least 20%, or at least 10% relative to theinflammation in an animal in not administered said antibodies.

Humanized antibodies of the invention can also be used to prevent therejection of transplants.

TABLE 4A Antibodies for Inflammatory Diseases and Autoimmune Diseasesthat can be used in combination with the antibodies of the invention.Antibody Target Name Antigen Product Type Isotype Sponsors Indication5G1.1 Complement Humanised IgG Alexion Pharm Inc Rheumatoid (C5)Arthritis 5G1.1 Complement Humanised IgG Alexion Pharm Inc SLE (C5)5G1.1 Complement Humanised IgG Alexion Pharm Inc Nephritis (C5) 5G1.1-SCComplement Humanised ScFv Alexion Pharm Inc Cardiopulmano (C5) Bypass5G1.1-SC Complement Humanised ScFv Alexion Pharm Inc Myocardial (C5)Infarction 5G1.1-SC Complement Humanised ScFv Alexion Pharm IncAngioplasty (C5) ABX- CBL Human Abgenix Inc GvHD CBL ™ ABX- CD147 MurineIgG Abgenix Inc Allograft rejection CBL ™ ABX- IL-8 Human IgG2 AbgenixInc Psoriasis IL8 ™ ANTEGREN ™ VLA-4 Humanised IgG Athena/Elan MultipleSclerosis Anti- CD11a Humanised IgG1 Genentech Psoriasis CD11a Inc/XomaAnti-CD18 CD18 Humanised Fab′2 Genentech Inc Myocardial infarctionAnti-LFA1 CD18 Murine Fab′2 Pasteur-Merieux/ Allograft rejectionImmunotech ANTOVA ™ CD40L Humanised IgG Biogen Allograft rejectionANTOVA ™ CD40L Humanised IgG Biogen SLE BTI-322 CD2 Rat IgG MedimmuneInc GvHD, Psoriasis CDP571 TNF-alpha Humanised IgG4 Celltech Crohn'sCDP571 TNF-alpha Humanised IgG4 Celltech Rheumatoid Arthritis CDP850E-selectin Humanised Celltech Psoriasis CORSEVINM ™ Fact VII ChimericCentocor Anticoagulant D2E7 TNF-alpha Human Abbott Rheumatoid ArthritisHu23F2G CD11/18 Humanised ICOS Pharm Inc Multiple Sclerosis Hu23F2GCD11/18 Humanised IgG ICOS Pharm Inc Stroke IC14 CD14 ICOS Pharm IncToxic shock ICM3 ICAM-3 Humanised ICOS Pharm Inc Psoriasis IDEC-114 ™CD80 Primatised IDEC Psoriasis Pharm/Mitsubishi IDEC-131 ™ CD40LHumanised IDEC Pharm/Eisai SLE IDEC-131 ™ CD40L Humanised IDECPharm/Eisai Multiple Sclerosis IDEC-151 ™ CD4 Primatised IgG1 IDECRheumatoid Pharm/GlaxoSmith Arthritis Kline IDEC-152 ™ CD23 PrimatisedIDEC Pharm Asthma/Allergy Infliximab TNF-alpha Chimeric IgG1 CentocorRheumatoid Arthritis Infliximab TNF-alpha Chimeric IgG1 Centocor Crohn'sLDP-01 ™ beta2- Humanised IgG Millennium Inc Stroke integrin (LeukoSiteInc.) LDP-01 ™ beta2- Humanised IgG Millennium Inc Allograft rejectionintegrin (LeukoSite Inc.) LDP-02 ™ alpha4beta7 Humanised Millennium IncUlcerative Colitis (LeukoSite Inc.) MAK-195F ™ TNF alpha Murine Fab′2Knoll Pharm, BASF Toxic shock MDX-33 ™ CD64 (FcR) Human Medarex/CenteonAutoimmune haematogical disorders MDX-CD4 ™ CD4 Human IgG Medarex/Eisai/Rheumatoid Genmab Arthritis MEDI-507 ™ CD2 Humanised Medimmune IncPsoriasis MEDI-507 ™ CD2 Humanised Medimmune Inc GvHD OKT4A ® CD4Humanised IgG Ortho Biotech Allograft rejection OrthoClone CD4 HumanisedIgG Ortho Biotech Autoimmune OKT4A ® disease Orthoclone/ CD3 MurinemIgG2a Ortho Biotech Allograft rejection anti-CD3 OKT3 ® RepPro/gpIIbIIIa Chimeric Fab Centocor/Lilly Complications of Abciximabcoronary angioplasty rhuMab- IgE Humanised IgG1 Genentech/Novartis/Asthma/Allergy E25 ™ Tanox Biosystems SB-240563 IL5 HumanisedGlaxoSmithKline Asthma/Allergy SB-240683 IL-4 Humanised GlaxoSmithKlineAsthma/Allergy SCH55700 IL-5 Humanised Celltech/Schering Asthma/AllergySimulect CD25 Chimeric IgG1 Novartis Pharm Allograft rejection SMART CD3Humanised Protein Design Lab Autoimmune a-CD3 ® disease SMART CD3Humanised Protein Design Lab Allograft rejection a-CD3 ® SMART CD3Humanised IgG Protein Design Lab Psoriasis a-CD3 ® Zenapax ® CD25Humanised IgG1 Protein Design Allograft rejection Lab/Hoffman- La Roche

TABLE 4B Antibodies and Fc fusion proteins for Autoimmune DisordersAntibody Indication Target Antigen ABX-RB2 ™ antibody to CBL antigen onT cells, B cells and NK cells fully human antibody from the XenomouseIL1-ra rheumatoid arthritis recombinant anti-inflammatory proteinsTNF-RI chronic inflammatory disease soluble tumor necrosis factor a -rheumatoid arthritis receptor type I blocks TNF action 5c8 (Anti CD-40Phase II trials were halted in Oct. CD-40 ligand antibody) 99 examine“adverse events” IDEC 131 ™ systemic lupus erythyematous anti CD40 (SLE)humanized IDEC 151 ™ rheumatoid arthritis primatized; anti-CD4 IDEC152 ™ Asthma primatized; anti-CD23 IDEC 114 ™ Psoriasis primatizedanti-CD80 MEDI-507 ™ rheumatoid arthritis; multiple anti-CD2 sclerosisCrohn's disease psoriasis LDP-02 (anti-b7 inflammatory bowel diseasea4b7 integrin receptor on white mAb) ™ Crohn's disease blood cells(leukocytes) ulcerative colitis SMART ® Anti- autoimmune disordersAnti-Gamma Interferon Gamma Interferon antibody Verteportin rheumatoidarthritis VERTEPORTIN ™ THALOMID ® leprosy - approved for marketinhibitor of tumor necrosis factor (thalidomide) Chron's disease alpha(TNF alpha) rheumatoid arthritis SelCIDs (selective highly specificcytokine inhibitory inhibitors of phosphodiesterase drugs) type 4 enzyme(PDE-4) increases levels of cAMP (cyclic adenosine monophosphate)activates protein kinase A (PKA) blocks transcription factor NK-kBprevents transcription of TNF-a gene decreases production of TNF-a IMiDsgeneral autoimmune disorders structural analogues of (immunomodulatorythalidomideinhibit TNF-a drugs) MDX-33 ™ blood disorders caused bymonoclonal antibody against FcRI autoimmune reactions receptorsIdiopathic Thrombocytopenia Purpurea (ITP) autoimmune hemolytic anemiaMDX-CD4 ™ treat rheumatoid arthritis and monoclonal antibody against CD4other autoimmunity receptor molecule VX-497 ™ autoimmune disordersinhibitor of inosine multiple sclerosis monophosphate dehydrogenaserheumatoid arthritis (enzyme needed to make new inflammatory boweldisease RNA and DNA lupus used in production of nucleotides psoriasisneeded for lymphocyte proliferation) VX-740 ™ rheumatoid arthritisinhibitor of ICE interleukin-1 beta (converting enzyme controls pathwaysleading to aggressive immune response regulates cytokines) VX-745 ™specific to inflammation inhibitor of P38MAP kinase involved in chemicalsignaling of mitogen activated protein kinase immune response onset andprogression of inflammation ENBREL ® targets TNF (tumor necrosis(etanercept) factor) IL-8 fully human MAB against IL-8 (interleukin 8)(blocks IL-8 blocks inflammatory response) 5G1.1 rheumatoid arthritis aC5 complement inhibitor pemphigoid (dangerous skin rash) psoriasis lupusAPOGEN ® MP4 recombinant antigen selectively destroys disease associatedT-cells induces apoptosis T-cells eliminated by programmed cell death nolonger attack body's own cells specific apogens target specific T- cells

6.3.4 Allergy

The invention provides methods for treating or preventing anIgE-mediated and or FcεRI mediated allergic disorder in a subject inneed thereof, comprising administering to said subject a therapeuticallyeffective amount of the agonistic antibodies or fragments thereof of theinvention. Although not intending to be bound by a particular mechanismof action, antibodies of the invention are useful in inhibitingFcεRI-induced mast cell activation, which contributes to acute and latephase allergic responses (Metcalfe D. et al. 1997, Physiol. Rev.77:1033; incorporated by reference in its entirety). Preferably, theagonistic antibodies of the invention have enhanced therapeutic efficacyand/or reduced side effects in comparison with the conventional methodsused in the art for the treatment and/or prevention of IgE mediatedallergic disorders. Conventional methods for the treatment and/orprevention of IgE mediated allergic disorders include, but are notlimited to, anti-inflammatory drugs (e.g., oral and inhaledcorticosteroids for asthma), antihistamines (e.g., for allergic rhinitisand atopic dermatitis), cysteinyl leukotrienes (e.g., for the treatmentof asthma); anti-IgE antibodies; and specific immunotherapy ordesensitization.

Examples of IgE-mediated allergic responses include, but are not limitedto, asthma, allergic rhinitis, gastrointestinal allergies, eosinophilia,conjunctivitis, atopic dermatitis, urticaria, anaphylaxis, or golmerularnephritis.

The invention encompasses molecules, e.g., immunoglobulins, engineeredto form complexes with FcεRI and human FcγRIIB, i.e., specifically bindFcεRI and human FcγRIIB. Preferably, such molecules have therapeuticefficacy in IgE and FcεRI-mediated disorders. Although not intending tobe bound by a particular mechanism of action, the therapeutic efficacyof these engineered molecules is, in part, due to their ability toinhibit mast cell and basophil function.

In a specific embodiment, molecules that specifically bind FcεRI andhuman FcγRIIB are chimeric fusion proteins comprising a binding site forFcεRI and a binding site for FcγRIIB. Such molecules may be engineeredin accordance with standard recombinant DNA methodologies known to oneskilled in the art. In a preferred specific embodiment, a chimericfusion protein for use in the methods of the invention comprises anF(ab′) single chain of an anti-FcγRIIB monoclonal antibody of theinvention fused to a region used as a bridge to link the huFcε to theC-terminal region of the F(ab′) single chain of the anti-FcγRIIBmonoclonal antibody. One exemplary chimeric fusion protein for use inthe methods of the invention comprises the following: V_(L)/C_(H)(FcγRIIB)-hinge-V_(H)/C_(H) (FcγRIIB)-LINKER-C_(H)ε2-C_(H)ε3-C_(H)ε4.The linker for the chimeric molecules may be five, ten, preferably,fifteen amino acids in length. The length of the linker may vary toprovide optimal binding of the molecule to both FcγRIIB and FcεRI. In aspecific embodiment, the linker is a 15 amino acid linker, consisting ofthe sequence: (Gly₄Ser)₃. Although not intending to be bound by aparticular mechanism of action, the flexible peptide linker facilitateschain pairing and minimizes possible refolding and it will also allowthe chimeric molecule to reach the two receptors, i.e., FcγRIIB andFcεRI on the cells and cross-link them. Preferably, the chimericmolecule is cloned into a mammalian expression vector, e.g., pCI-neo,with a compatible promoter, e.g., cytomegalovirus promoter. The fusionprotein prepared in accordance with the methods of the invention willcontain the binding site for FcεRI (CHε2CHε3) and for FcγRIIB (VL/CL,-hinge-VH/CH). The nucleic acid encoding the fusion protein prepared inaccordance with the methods of the invention is preferably transfectedinto 293 cells and the secreted protein is purified using common methodsknown in the art.

Binding of the chimeric molecules to both human FcεRI and FcγRIIB may beassessed using common methods known to one skilled in the art fordetermining binding to an FcγR. Preferably, the chimeric molecules ofthe invention have therapeutic efficacy in treating IgE mediateddisorders, for example, by inhibiting antigen-driven degranulation andinhibition of cell activation. The efficacy of the chimeric molecules ofthe invention in blocking IgE driven FcεRI-mediated mast celldegranulation may be determined in transgenic mice, which have beenengineered to express the human FcεRα and human FcγRIIB, prior to theiruse in humans.

The invention provides the use of bispecific antibodies for thetreatment and/or prevention of IgE-mediated and/or FcεRI-mediatedallergic disorders. A bispecific antibody (BsAb) binds to two differentepitopes usually on distinct antigens. BsAbs have potential clinicalutility and they have been used to target viruses, virally infectedcells and bacterial pathogens as well as to deliver thrombolitic agentsto blood clots (Cao Y., 1998 Bioconj. Chem. 9: 635-644; Koelemij et al.,1999, J. Immunother., 22, 514-524; Segal et al., Curr. Opin. Immunol.,11, 558-562). The technology for the production of BsIgG and otherrelated bispecific molecules is available (see, e.g., Carter et al.,2001 J. of Immunol. Methods, 248, 7-15; Segal et al., 2001, J. ofImmunol. Methods, 248, 7-15, which are incorporated herein by referencein their entirety). The instant invention provides bispecific antibodiescontaining one F(ab′) of the anti-FcγRIIB antibody and one F(ab′) of anavailable monoclonal anti-huIgE antibody which aggregates two receptors,FcγRIIB and FcεRI, on the surface of the same cell. Any methodologyknown in the art and disclosed herein may be employed to generatebispecific antibodies for use in the methods of the invention. In aspecific embodiment, the BsAbs will be produced by chemicallycross-linking F(ab′) fragments of an anti-FcγRIIB antibody and ananti-huIgE antibody as described previously (see, e.g., Glennie et al.,1995, Tumor Immunobiology, Oxford University press, Oxford, p. 225;which is incorporated herein by reference in its entirety). The F(ab′)fragments may be produced by limited proteolysis with pepsin and reducedwith mercaptoethanol amine to provide Fab′ fragments with freehinge-region sulfhydryl (SH) groups. The SH group on one of the Fab′(SH) fragments may be alkylated with excess O-phenylenedimaleimide(O-PDM) to provide a free maleimide group (mal). The two preparationsFab′(mal) and Fab′(SH) may be combined at an appropriate ratio,preferably, 1:1 to generate heterodimeric constructs. The BsAbs can bepurified by size exclusion chromatography and characterized by HPLCusing methods known to one skilled in thr art.

In particular, the invention encompasses bispecific antibodiescomprising a first heavy chain-light chain pair that binds FcγRIIB withgreater affinity than said heavy chain-light chain pair binds FcγRIIA,and a second heavy chain-light chain pair that binds IgE receptor, withthe provision that said first heavy chain-light chain pair binds FcγRIIBfirst. The bispecific antibodies of the invention can be engineeredusing standard techniques known in the art to ensure that the binding toFcγRIIB precedes the binding to the IgE receptor. It will be understoodto one skilled in the art to engineer the bispecific antibodies, forexample, such that said bispecific antibodies bind FcγRIIB with greateraffinity than said antibodies bind IgE receptor. Additionally, thebispecific antibodies can be engineered by techniques known in the art,such that the hinge size of the antibody can be increased in length, forexample, by adding linkers, to provide the bispecific antibodies withflexibility to bind the IgE receptor and FcγRIIB receptor on the samecell.

The humanized antibodies of the invention can also be used incombination with other therapeutic antibodies or drugs known in the artfor the treatment or prevention of IgE-mediated allergic disorders. Forexample, the antibodies of the invention can be used in combination withany of the following: azelastine, Astelin, beclomethasone dipropionateinhaler, Vanceril, beclomethasone dipropionate nasal inhaler/spray,Vancenase, Beconase budesonide nasal inhaler/spray, Rhinocortcetirizine, Zyrtec chlorpheniramine, pseudoephedrine, Deconamine,Sudafed, cromolyn, Nasalcrom, Intal, Opticrom, desloratadine, Clarinex,fexofenadine and pseudoephedrine, Allegra-D, fexofenadine, Allegraflunisolide nasal spray, Nasalide fluticasone propionate nasalinhaler/spray, Flonase fluticasone propionate oral inhaler, Flovent,hydroxyzine, Vistaril, Ataraxloratadine, pseudoephedrine, Claritin-D,loratadine, Claritin, prednisolone, Prednisolone, Pediapred Oral Liquid,Medrol prednisone, Deltasone, Liquid Predsalmeterol, Sereventtriamcinolone acetonide inhaler, Azmacort triamcinolone acetonide nasalinhaler/spray, Nasacort, or NasacortAQ. Antibodies of the invention canbe used in combination with cytosine-guanine dinucleotides (“CpG”)-basedproducts that have been developed (Coley Pharmaceuticals) or arecurrently being developed as activators of innate and acquired immuneresponses. For example, the invention encompasses the use of CpG 7909,CpG 8916, CpG 8954 (Coley Pharmaceuticals) in the methods andcompositions of the invention for the treatment and/or prevention ofIgE-mediated allergic disorders (See also Weeratna et al., 2001, FEMSImmunol Med. Microbiol., 32(1):65-71, which is incorporated herein byreference).

The invention encompasses the use of the humanized antibodies of theinvention in combination with any therapeutic antibodies known in theart for the treatment of allergy disorders, e.g., Xolair™ (Omalizumab;Genentech); rhuMAB-E25 (BioWorld Today, Nov. 10, 1998, p. 1; Genentech);CGP-51901 (humanized anti-IgE antibody), etc.

Additionally, the invention encompasses the use of the humanizedantibodies of the invention in combination with other compositions knownin the art for the treatment of allergy disorders. In particular methodsand compositions disclosed in Carson et al. (U.S. Pat. No. 6,426,336;U.S. Patent Application Publication Nos. 2002/0035109 A1 and2002/0010343, all of which are incorporated herein by reference in itsentirety).

6.3.5 Immunomodulatory Agents and Anti-Inflammatory Agents

The method of the present invention provides methods of treatment forautoimmune diseases and inflammatory diseases comprising administrationof the antibodies of the present invention in conjunction with othertreatment agents. Examples of immunomodulatory agents include, but arenot limited to, methothrexate, ENBREL®, REMICADE™, HUMIRA®, leflunomide,cyclophosphamide, cyclosporine A, and macrolide antibiotics (e.g., FK506(tacrolimus)), methylprednisolone (MP), corticosteroids, steriods,mycophenolate mofetil, rapamycin (sirolimus), mizoribine,deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), Tcell receptor modulators, and cytokine receptor modulators.

Anti-inflammatory agents have exhibited success in treatment ofinflammatory and autoimmune disorders and are now a common and astandard treatment for such disorders. Any anti-inflammatory agentwell-known to one of skill in the art can be used in the methods of theinvention. Non-limiting examples of anti-inflammatory agents includenon-steroidal anti-inflammatory drugs (NSAIDs), steroidalanti-inflammatory drugs, beta-agonists, anticholingeric agents, andmethyl xanthines. Examples of NSAIDs include, but are not limited to,aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac (VOLTAREN™),etodolac (LODINE™) fenoprofen (NALFON™), indomethacin (INDOCINT™),ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumentone (RELAFEN™),sulindac (CLINORILT™), tolmentin (TOLECTINT™), rofecoxib (VIOXX™),naproxen (ALEVE™, NAPROSYN™), ketoprofen (ACTRON™) and nabumetone(RELAFEN™). Such NSAIDs function by inhibiting a cyclooxygenase enzyme(e.g., COX-1 and/or COX-2). Examples of steroidal anti-inflammatorydrugs include, but are not limited to, glucocorticoids, dexamethasone(DECADRON™) cortisone, hydrocortisone, prednisone (DELTASONE™),prednisolone, triamcinolone, azulfidine, and eicosanoids such asprostaglandins, thromboxanes, and leukotrienes.

6.3.6 Anti-Cancer Agents and Therapeutic Antibodies

In a specific embodiment, the methods of the invention encompass theadministration of one or more angiogenesis inhibitors such as, but notlimited to: Angiostatin (plasminogen fragment); antiangiogenicantithrombin III; Angiozyme; ABT-627; Bay 12-9566; Benefin; Bevacizumab;BMS-275291; cartilage-derived inhibitor (CDI); CAI; CD59 complementfragment; CEP-7055; Col 3; Combretastatin A-4; Endostatin (collagenXVIII fragment); EGFr blockers/inhibitors (Iressa®, Tarceva®, Erbitux®,and ABX-EGF) Fibronectin fragment; Gro-beta; Halofuginone; Heparinases;Heparin hexasaccharide fragment; HMV833; Human chorionic gonadotropin(hCG); IM-862; Interferon alpha/beta/gamma; Interferon inducible protein(IP-10); Interleukin-12; Kringle 5 (plasminogen fragment); Marimastat;Metalloproteinase inhibitors (TIMPs); 2-Methoxyestradiol; MMI 270 (CGS27023A); MoAb IMC-1C11; Neovastat; NM-3; Panzem; PI-88; Placentalribonuclease inhibitor; Plasminogen activator inhibitor; Plateletfactor-4 (PF4); Prinomastat; Prolactin 16 kD fragment;Proliferin-related protein (PRP); PTK 787/ZK 222594; Retinoids;Solimastat; Squalamine; SS 3304; SU 5416; SU6668; SU11248;Tetrahydrocortisol-S; tetrathiomolybdate; thalidomide; Thrombospondin-1(TSP-1); TNP-470; Transforming growth factor-beta (TGF-b);Vasculostatin; Vasostatin (calreticulin fragment); ZD6126; ZD 6474;farnesyl transferase inhibitors (FTI); and bisphosphonates.

Anti-cancer agents that can be used in combination with antibodies ofthe invention in the various embodiments of the invention, includingpharmaceutical compositions and dosage forms and kits of the invention,include, but are not limited to: acivicin; aclarubicin; acodazolehydrochloride; acronine; adozelesin; aldesleukin; altretamine;ambomycin; ametantrone acetate; aminoglutethimide; amsacrine;anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa;azotomycin; batimastat; benzodepa; bicalutamide; bisantrenehydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate;brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interleukin II (including recombinant interleukin II, orrIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1;interferon alfa-n3; interferon beta-I a; interferon gamma-I b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride. Other anti-cancer drugs include,but are not limited to: 20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol; dioxamycin; diphenyl spiromustine; docetaxel; docosanol;dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA;ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene;emitefur; epirubicin; epristeride; estramustine analogue; estrogenagonists; estrogen antagonists; etanidazole; etoposide phosphate;exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. Preferred additional anti-cancer drugs are 5-fluorouraciland leucovorin.

Examples of therapeutic antibodies that can be used in methods of theinvention include, but are not limited to, HERCEPTIN® (Trastuzumab)(Genentech, South San Francisco, Calif.) which is a humanized anti-HER2monoclonal antibody for the treatment of patients with metastatic breastcancer; REOPRO® (abciximab) (Centocor) which is an anti-glycoproteinIIb/IIIa receptor on the platelets for the prevention of clot formation;ZENAPAX® (daclizumab) (Roche Pharmaceuticals, Switzerland) which is animmunosuppressive, humanized anti-CD25 monoclonal antibody for theprevention of acute renal allograft rejection; PANOREX™ which is amurine anti-17-IA cell surface antigen IgG2a antibody (GlaxoWellcome/Centocor); BEC2 which is a murine anti-idiotype (GD3 epitope)IgG antibody (ImClone System); IMC-C225 which is a chimeric anti-EGFRIgGantibody (ImClone System); VITAXIN™ which is a humanized anti-αVβ3integrin antibody (Applied Molecular Evolution/MedImmune); Campath1H/LDP-03 which is a humanized anti CD52 IgG1 antibody (Leukosite);Smart M195 which is a humanized anti-CD33 IgG antibody (Protein DesignLab/Kanebo); RITUXAN™ (rituximab) which is a chimeric anti-CD20 IgG1antibody (IDEC Pharm/Genentech, Roche/Zettyaku); LYMPHOCIDE™(epratuzumab) which is a humanized anti-CD22 IgG antibody(Immunomedics); ICM3 which is a humanized anti-ICAM3 antibody (ICOSPharm); IDEC-114 which is a primatied anti-CD80 antibody (IDECPharm/Mitsubishi); ZEVALIN™ which is a radiolabelled murine anti-CD20antibody (IDEC/Schering AG); IDEC-131 which is a humanized anti-CD40Lantibody (IDEC/Eisai); IDEC-151 which is a primatized anti-CD4 antibody(IDEC); IDEC-152 which is a primatized anti-CD23 antibody(IDEC/Seikagaku); SMART anti-CD3 which is a humanized anti-CD3 IgG(Protein Design Lab); 5G1.1 which is a humanized anti-complement factor5 (C5) antibody (Alexion Pharm); D2E7 which is a humanized anti-TNF-αantibody (CAT/BASF); CDP870 which is a humanized anti-TNF-α Fab fragment(Celltech); IDEC-151 which is a primatized anti-CD4 IgG1 antibody (IDECPharm/SmithKline Beecham); MDX-CD4 which is a human anti-CD4 IgGantibody (Medarex/Eisai/Genmab); CDP571 which is a humanized anti-TNF-αIgG4 antibody (Celltech); LDP-02 which is a humanized anti-α4β7 antibody(LeukoSite/Genentech); OrthoClone OKT4A which is a humanized anti-CD4IgG antibody (Ortho Biotech); ANTOVA™ which is a humanized anti-CD40LIgG antibody (Biogen); ANTEGREN™ which is a humanized anti-VLA-4 IgGantibody (Elan); and CAT-152 which is a human anti-TGF-β₂ antibody(Cambridge Ab Tech).

Other examples of therapeutic antibodies that can be used in combinationwith the antibodies of the invention are presented in Table 5.

TABLE 5 Monoclonal antibodies for Cancer Therapy that can be used incombination with the antibodies of the invention. Company ProductDisease Target Abgenix ABX-EGF Cancer EGF receptor AltaRex OvaRex ®ovarian cancer tumor antigen CA125 BravaRex ® metastatic tumor antigenMUC1 cancers Antisoma Theragyn ovarian cancer PEM antigen(pemtumomabytrrium- 90) THEREX ® breast cancer PEM antigen Boehringerblvatuzumab head & neck CD44 Ingelheim cancer Centocor/J&J PANOREX ®Colorectal 17-1A cancer REOPRO ® PTCA gp IIIb/IIIa REOPRO ® Acute MI gpIIIb/IIIa REOPRO ® Ischemic stroke gp IIIb/IIIa Corixa Bexocar NHL CD20CRC Technology MAb, idiotypic 105AD7 colorectal cancer gp72 vaccineCrucell Anti-EpCAM cancer Ep-CAM Cytoclonal MAb, lung cancer non-smallcell NA lung cancer Genentech HERCEPTIN ® metastatic breast HER-2 cancerHERCEPTIN ® early stage HER-2 breast cancer RITUXAN ® Relapsed/refractCD20 ory low-grade or follicular NHL RITUXAN ® intermediate & CD20high-grade NHL MAb-VEGF NSCLC, VEGF metastatic MAb-VEGF Colorectal VEGFcancer, metastatic AMD Fab age-related CD18 macular degeneration E-26(2^(nd) gen. IgE) allergic asthma IgE & rhinitis IDEC ZEVALIN ® lowgrade of CD20 (RITUXAN ® +yttrium- follicular, 90) relapsed orrefractory, CD20-positive, B-cell NHL and Rituximab- refractory NHLImClone Cetuximab + innotecan refractory EGF receptor colorectalcarcinoma Cetuximab + cisplatin & newly diagnosed EGF receptor radiationor recurrent head & neck cancer Cetuximab + newly diagnosed EGF receptorgemcitabine metastatic pancreatic carcinoma Cetuximab + cisplatin +recurrent or EGF receptor 5FU or TAXOL ® metastatic head & neck cancerCetuximab + newly diagnosed EGF receptor carboplatin + paclitaxelnon-small cell lung carcinoma Cetuximab + cisplatin head & neck EGFreceptor cancer (extensive incurable local- regional disease & distantmetasteses) Cetuximab + radiation locally advanced EGF receptor head &neck carcinoma BEC2 + Bacillus small cell lung mimics gangliosideCalmette Guerin carcinoma GD3 BEC2 + Bacillus melanoma mimicsganglioside Calmette Guerin GD3 IMC-1C11 colorectal cancer VEGF-receptorwith liver metasteses ImmonoGen nuC242-DM1 Colorectal, nuC242 gastric,and pancreatic cancer ImmunoMedics LymphoCide Non-Hodgkins CD22 lymphomaLymphoCide Y-90 Non-Hodgkins CD22 lymphoma CEA-Cide metastatic solid CEAtumors CEA-Cide Y-90 metastatic solid CEA tumors CEA-Scan (Tc-99m-colorectal cancer CEA labeled arcitumomab) (radioimaging) CEA-Scan(Tc-99m- Breast cancer CEA labeled arcitumomab) (radioimaging) CEA-Scan(Tc-99m- lung cancer CEA labeled arcitumomab) (radioimaging) CEA-Scan(Tc-99m- intraoperative CEA labeled arcitumomab) tumors (radio imaging)LEUKOSCAN ® (Tc- soft tissue CEA 99m-labeled sulesomab) infection(radioimaging) LYMPHOSCAN ® (Tc- lymphomas CD22 99m-labeled)(radioimaging) AFP-Scan (Tc-99m- liver 7 gem-cell AFP labeled) cancers(radioimaging) Intracel HumaRAD ®-HN (+ head & neck NA yttrium-90)cancer HumaSPECT ® colorectal NA imaging Medarex MDX-101 ® (CTLA-4)Prostate and CTLA-4 other cancers MDX-210 ® (her-2 Prostate cancer HER-2overexpression) MDX-210 ®/MAK Cancer HER-2 MedImmune Vitaxin Cancer αvβ₃Merck KGaA MAb 425 Various cancers EGF receptor IS-IL-2 Various cancersEp-CAM Millennium CAMPATH ® chronic CD52 (alemtuzumab) lymphocyticleukemia NeoRx CD20-streptavidin (+ Non-Hodgkins CD20 biotin-yttrium 90)lymphoma Avidicin (albumin + metastatic NA NRLU13) cancer PeregrineOncolym (+ iodine-131) Non-Hodgkins HLA-DR 10 beta lymphoma COTARA ® (+iodine- unresectable DNA-associated 131) malignant proteins gliomaPharmacia C215 (+ staphylococcal pancreatic NA Corporation enterotoxin)cancer MAb, lung/kidney lung & kidney NA cancer cancer nacolomabtafenatox colon & NA (C242 + staphylococcal pancreatic enterotoxin)cancer Protein Design Nuvion T cell CD3 Labs malignancies SMART M195 ®AML CD33 SMART 1D10 ® NHL HLA-DR antigen Titan CEAVac colorectal CEAcancer, advanced TriGem metastatic GD2-ganglioside melanoma & small celllung cancer TriAb metastatic breast MUC-1 cancer Trilex CEAVaccolorectal CEA cancer, advanced TriGem metastatic GD2-gangliosidemelanoma & small cell lung cancer TriAb metastatic breast MUC-1 cancerViventia Biotech NovoMAb-G2 Non-Hodgkins NA radiolabeled lymphomaMonopharm C colorectal & SK-1 antigen pancreatic carcinoma GlioMAb-H (+gelonin gliorna, NA toxin) melanoma & neuroblastoma Xoma RITUXAN ®Relapsed/refractory CD20 low-grade or follicular NHL RITUXAN ®intermediate & CD20 high-grade NHL ING-1 adenomcarcinoma Ep-CAM

6.3.7 Vaccine Therapy and Prophylaxis

The invention provides a method for enhancing an immune response to avaccine composition in a subject, said method comprising administeringto said subject a humanized antibody of the invention or a fragmentthereof that specifically binds FcγRIIB with greater affinity than saidantibody or a fragment thereof binds FcγRIIA, and a vaccine composition,wherein said antibody or a fragment thereof enhances the immune responseto said vaccine composition. In one particular embodiment, said antibodyor a fragment thereof enhances the immune response to said vaccinecomposition by enhancing antigen presentation/and or antigen processingof the antigen to which the vaccine is directed at. Any vaccinecomposition known in the art is useful in combination with theantibodies or fragments thereof of the invention.

Although not intending to be bound by a particular mechanism of action,the antibodies of the invention may block activation of FcγRIIB that isexpressed on certain populations and/or types of dendritic cells andthus enhance the activity of such dendritic cells during activevaccination. This enhanced dendritic cell activity may thus result in anenhanced or better response to prophylatic or therapeutic vaccination.

In one embodiment, the invention encompasses the use of the humanizedantibodies of the invention in combination with any cancer vaccine knownin the art, e.g., Canvaxin™ (Cancer Vax, Corporation, melanoma and coloncancer); Oncophage (HSPPC-96; Antigenics; metastatic melanoma);HER-2/neu cancer vaccine, etc. The cancer vaccines used in the methodsand compositions of the invention can be, for example, antigen-specificvaccines, anti-idiotypic vaccines, dendritic cell vaccines, or DNAvaccines. In other embodiments, the invention encompasses use of theantibodies of the invention with vaccines against EGFRviii, CD44 splicevariants, and PSMA. The invention encompasses the use of the antibodiesof the invention with cell-based vaccines as described by Segal et al.(U.S. Pat. No. 6,403,080, which is incorporated herein by reference inits entirety). The cell based vaccines used in combination with theantibodies of the invention can be either autologous or allogeneic.Briefly, the cancer-based vaccines as described by Segal et al. arebased on Opsonokine™ product by Genitrix, LLC. Opsonokines™ aregenetically engineered cytokines that, when mixed with tumor cells,automatically attach to the surface of the cells. When the “decorated”cells are administered as a vaccine, the cytokine on the cells activatescritical antigen presenting cells in the recipient, while also allowingthe antigen presenting cells to ingest the tumor cells. The antigenpresenting cells are then able to instruct “killer” T cells to find anddestroy similar tumor cells throughout the body. Thus, the Opsonokine™product converts the tumor cells into a potent anti-tumorimmunotherapeutic.

In one embodiment, the invention encompasses the use of the humanizedantibodies of the invention in combination with any allergy vaccineknown in the art. The humanized antibodies of the invention, can beused, for example, in combination with recombinant hybrid moleculescoding for the major timothy grass pollen allergens used for vaccinationagainst grass pollen allergy, as described by Linhart et al. (2000,FASEB Journal, 16(10):1301-3, which is incorporated by reference). Inaddition, the humanized antibodies of the invention can be used incombination with DNA-based vaccinations described by Horner et al.(2002, Allergy, 57 Suppl, 72:24-9, which is incorporated by reference).Antibodies of the invention can be used in combination with BacilleClamett-Guerin (“BCG”) vaccination as described by Choi et al. (2002,Ann. Allergy Asthma Immunology, 88(6): 584-91) and Barlan et al. (2002,Journal Asthma, 39(3):239-46), both of which are incorporated herein byreference in entirety, to downregulate IgE secretion. The humanizedantibodies of the invention are useful in treating food allergies. Inparticular the humanized antibodies of the invention can be used incombination with vaccines or other immunotherapies known in the art (seeHourihane et al., 2002, Curr. Opin. Allergy Clin. Immunol. 2(3):227-31)for the treatment of peanut allergies.

The methods and compositions of the invention can be used in combinationwith vaccines, in which immunity for the antigen(s) is desired. Suchantigens may be any antigen known in the art. The humanized antibodiesof the invention can be used to enhance an immune response, for example,to infectious agents, diseased or abnormal cells such as, but notlimited to, bacteria (e.g., gram positive bacteria, gram negativebacteria, aerobic bacteria, Spirochetes, Mycobacteria, Rickettsias,Chlamydias, etc.), parasites, fungi (e.g., Candida albicans,Aspergillus, etc.), viruses (e.g., DNA viruses, RNA viruses, etc.), ortumors. Viral infections include, but are not limited to, humanimmunodeficiency virus (HIV); hepatitis A virus, hepatitis B virus,hepatitis C virus, hepatitis D virus, or other hepatitis viruses;cytomagaloviruses, herpes simplex virus-1 (-2, -3, -4, -5, -6), humanpapilloma viruses; Respiratory syncytial virus (RSV), Parainfluenzavirus (PIV), Epstein Barr virus, human metapneumovirus (HMPV), influenzavirus, Severe Acute Respiratory Syndrome (SARS) or any other viralinfections.

The invention encompasses methods and vaccine compositions comprisingcombinations of a humanized antibody of the invention, an antigen and acytokine. Preferably, the cytokine is IL-4, IL-10, or TGF-β.

The invention encompasses the use of the humanized antibodies of theinvention to enhance a humoral and/or cell mediated response against theantigen(s) of the vaccine composition. The invention further encompassesthe use of the humanized antibodies of the invention to either preventor treat a particular disorder, where an enhanced immune responseagainst a particular antigen or antigens is effective to treat orprevent the disease or disorder. Such diseases and disorders include,but are not limited to, viral infections, such as HIV, CMV, hepatitis,herpes virus, measles, etc., bacterial infections, fungal and parasiticinfections, cancers, and any other disease or disorder amenable totreatment or prevention by enhancing an immune response against aparticular antigen or antigens.

6.3.8 Breaking Tolerance to an Antigen

Certain cancers may be associated with an ability of the tumors tocircumvent an immune response against their antigens, i.e., tolerance tothese antigens exists. See Mapara et al., 2004, J. Clin. Oncol.22:1136-1151; incorporated by reference in its entirety. Accordingly, agoal in tumor immunotherapy is to break tolerance to tumor antigens inorder to induce an antitumor response. Eliciting an immune responseagainst a foreign antigen that is otherwise recognized by the host as a“self” antigen breaks tolerance to that antigen.

Thus, in certain embodiments, the invention provides a method forbreaking tolerance to an antigen in a patient by administering to apatient in need thereof (1) an antigen-antibody complex comprising theantigen and (2) a humanized antibody or fragment thereof thatspecifically binds the extracellular domain of human FcγRIIB and blocksthe Fc binding site of human FcγRIIB, thereby breaking tolerance in saidpatient to the antigen. The humanized antibody or fragment thereof canbe administered before, concurrently with, or after administration ofsaid antigen-antibody complex.

Antigen-presenting cells, such as dendritic cells, coexpress activatingand inhibitory Fc gamma receptors. Without being bound by theory, whenantibodies that block Fc binding to FcγRIIB are present, theantigen-antibody complexes comprising an antigen are primarily taken upby non-inhibitory receptors on antigen-presenting cells elicting animmune response to the antigen.

In certain embodiments, the antigen is an antigen that is associatedwith a cancer or a neoplastic disease. In another aspect, the antigen isspecific to a cancer cell or a neoplastic cell. The antigen can also bean antigen of a pathogen, such as, e.g., a virus, a bacterium, or aprotozoa. Representative antigens have been disclosed herein.

6.4 Compositions and Methods of Administering

The invention provides methods and pharmaceutical compositionscomprising the humanized antibodies of the invention. The invention alsoprovides methods of treatment, prophylaxis, and amelioration of one ormore symptoms associated with a disease, disorder or infection byadministering to a subject an effective amount of a fusion protein or aconjugated molecule of the invention, or a pharmaceutical compositioncomprising a fusion protein or conjugated molecules of the invention. Ina preferred aspect, an antibody or fusion protein or conjugatedmolecule, is substantially purified (i.e., substantially free fromsubstances that limit its effect or produce undesired side-effects). Ina specific embodiment, the subject is an animal, preferably a mammalsuch as non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.)and a primate (e.g., monkey such as, a cynomolgous monkey and a human).In a preferred embodiment, the subject is a human.

Various delivery systems are known and can be used to administer acomposition comprising humanized antibodies of the invention, e.g.,encapsulation in liposomes, microparticles, microcapsules, recombinantcells capable of expressing the antibody or fusion protein,receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol.Chem. 262:4429-4432; incorporated by reference in its entirety),construction of a nucleic acid as part of a retroviral or other vector,etc.

In some embodiments, the humanized antibodies of the invention areformulated in liposomes for targeted delivery of the antibodies of theinvention. Liposomes are vesicles comprised of concentrically orderedphopsholipid bilayers which encapsulate an aqueous phase. Liposomestypically comprise various types of lipids, phospholipids, and/orsurfactants. The components of liposomes are arranged in a bilayerconfiguration, similar to the lipid arrangement of biological membranes.Liposomes are particularly preferred delivery vehicles due, in part, totheir biocompatibility, low immunogenicity, and low toxicity. Methodsfor preparation of liposomes are known in the art and are encompassedwithin the invention, see, e.g., Epstein et al., 1985, Proc. Natl. Acad.Sci. USA, 82: 3688; Hwang et al., 1980 Proc. Natl. Acad. Sci. USA, 77:4030-4; U.S. Pat. Nos. 4,485,045 and 4,544,545; all of which areincorporated herein by reference in their entirety.

The invention also encompasses methods of preparing liposomes with aprolonged serum half-life, i.e., enhanced circulation time, such asthose disclosed in U.S. Pat. No. 5,013,556. Preferred liposomes used inthe methods of the invention are not rapidly cleared from circulation,i.e., are not taken up into the mononuclear phagocyte system (MPS). Theinvention encompasses sterically stabilized liposomes which are preparedusing common methods known to one skilled in the art. Although notintending to be bound by a particular mechanism of action, stericallystabilized liposomes contain lipid components with bulky and highlyflexible hydrophilic moieties, which reduces the unwanted reaction ofliposomes with serum proteins, reduces oposonization with serumcomponents and reduces recognition by MPS. Sterically stabilizedliposomes are preferably prepared using polyethylene glycol. Forpreparation of liposomes and sterically stabilized liposome, see, e.g.,Bendas et al., 2001 BioDrugs, 15(4): 215-224; Allen et al., 1987 FEBSLett. 223: 42-6; Klibanov et al., 1990 FEBS Lett., 268: 235-7; Blum etal., 1990, Biochim. Biophys. Acta., 1029: 91-7; Torchilin et al., 1996,J. Liposome Res. 6: 99-116; Litzinger et al., 1994, Biochim. Biophys.Acta, 1190: 99-107; Maruyama et al., 1991, Chem. Pharm. Bull., 39:1620-2; Klibanov et al., 1991, Biochim Biophys Acta, 1062; 142-8; Allenet al., 1994, Adv. Drug Deliv. Rev, 13: 285-309; all of which areincorporated herein by reference in their entirety. The invention alsoencompasses liposomes that are adapted for specific organ targeting,see, e.g., U.S. Pat. No. 4,544,545, or specific cell targeting, see,e.g., U.S. Patent Application Publication No. 2005/0074403. Particularlyuseful liposomes for use in the compositions and methods of theinvention can be generated by reverse phase evaporation method with alipid composition comprising phosphatidylcholine, cholesterol, and PEGderivatized phosphatidylethanolamine (PEG-PE). Liposomes are extrudedthrough filters of defined pore size to yield liposomes with the desireddiameter. In some embodiments, a fragment of an antibody of theinvention, e.g., F(ab′), may be conjugated to the liposomes usingpreviously described methods, see, e.g., Martin et al., 1982, J. Biol.Chem. 257: 286-288, which is incorporated herein by reference in itsentirety.

The humanized antibodies of the invention may also be formulated asimmunoliposomes. Immunoliposomes refer to a liposomal composition,wherein an antibody of the invention or a fragment thereof is linked,covalently or non-covalently to the liposomal surface. The chemistry oflinking an antibody to the liposomal surface is known in the art andencompassed within the invention, see, e.g., U.S. Pat. No. 6,787,153;Allen et al., 1995, Stealth Liposomes, Boca Rotan: CRC Press, 233-44;Hansen et al., 1995, Biochim. Biophys. Acta, 1239: 133-44; which areincorporated herein by reference in their entirety. In most preferredembodiments, immunoliposomes for use in the methods and compositions ofthe invention are further sterically stabilized. Preferably, thehumanized antibodies of the invention are linked covalently ornon-covalently to a hydrophobic anchor, which is stably rooted in thelipid bilayer of the liposome. Examples of hydrophobic anchors include,but are not limited to, phospholipids, e.g., phosoatidylethanolamine(PE), phospahtidylinositol (PI). To achieve a covalent linkage betweenan antibody and a hydrophobic anchor, any of the known biochemicalstrategies in the art may be used, see, e.g., J. Thomas August, ed.,1997, Gene Therapy: Advances in Pharmacology, Volume 40, Academic Press,San Diego, Calif., p. 399-435, which is incorporated herein by referencein its entirety. For example, a functional group on an antibody moleculemay react with an active group on a liposome associated hydrophobicanchor, e.g., an amino group of a lysine side chain on an antibody maybe coupled to liposome associated N-glutaryl-phosphatidylethanolamineactivated with water-soluble carbodiimide; or a thiol group of a reducedantibody can be coupled to liposomes via thiol reactive anchors, such aspyridylthiopropionyl-phosphatidylethanolamine See, e.g., Dietrich etal., 1996, Biochemistry, 35: 1100-1105; Loughrey et al., 1987, Biochim.Biophys. Acta, 901: 157-160; Martin et al., 1982, J. Biol. Chem. 257:286-288; Martin et al., 1981, Biochemistry, 20: 4429-38; all of whichare incorporated herein by reference in their entirety. Although notintending to be bound by a particular mechanism of action,immunoliposomal formulations comprising an antibody of the invention areparticularly effective as therapeutic agents, since they deliver theantibody to the cytoplasm of the target cell, i.e., the cell comprisingthe FcγRIIB receptor to which the antibody binds. The immunoliposomespreferably have an increased half-life in blood, specifically targetcells, and can be internalized into the cytoplasm of the target cellsthereby avoiding loss of the therapeutic agent or degradation by theendolysosomal pathway.

The invention encompasses immunoliposomes comprising a humanizedantibody of the invention or a fragment thereof. In some embodiments,the immunoliposomes further comprise one or more additional therapeuticagents, such as those disclosed herein.

The immunoliposomal compositions of the invention comprise one or morevesicle forming lipids, an antibody of the invention or a fragment orderivative thereof, and, optionally, a hydrophilic polymer. A vesicleforming lipid is preferably a lipid with two hydrocarbon chains, such asacyl chains and a polar head group. Examples of vesicle forming lipidsinclude phospholipids, e.g., phosphatidylcholine,phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol,sphingomyelin, and glycolipids, e.g., cerebrosides, gangliosides.Additional lipids useful in the formulations of the invention are knownto one skilled in the art and encompassed within the invention. In someembodiments, the immunoliposomal compositions further comprise ahydrophilic polymer, e.g., polyethylene glycol, and ganglioside GM1,which increases the serum half life of the liposome. Methods ofconjugating hydrophilic polymers to liposomes are well known in the artand encompassed within the invention. For a review of immunoliposomesand methods of preparing them, see, e.g., U.S. Patent ApplicationPublication No. 2003/0044407; PCT International Publication No. WO97/38731, Vingerhoeads et al., 1994, Immunomethods, 4: 259-72; Maruyama,2000, Biol. Pharm. Bull. 23(7): 791-799; Abra et al., 2002, Journal ofLiposome Research, 12(1&2): 1-3; Park, 2002, Bioscience Reports, 22(2):267-281; Bendas et al., 2001 BioDrugs, 14(4): 215-224, J. Thomas August,ed., 1997, Gene Therapy Advances in Pharmacology, Volume 40, AcademicPress, San Diego, Calif., p. 399-435, all of which are incorporatedherein by reference in their entireties.

Methods of administering a humanized antibody of the invention include,but are not limited to, parenteral administration (e.g., intradermal,intramuscular, intraperitoneal, intravenous and subcutaneous), epidural,and mucosal (e.g., intranasal and oral routes). In a specificembodiment, the antibodies of the invention are administeredintramuscularly, intravenously, or subcutaneously. The compositions maybe administered by any convenient route, for example, by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically active agents.Administration can be systemic or local. In addition, pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968; 5,985, 20; 5,985,309; 5,934,272; 5,874,064;5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, eachof which is incorporated herein by reference in its entirety.

The invention also provides that the humanized antibodies of theinvention are packaged in a hermetically sealed container, such as anampoule or sachette, indicating the quantity of antibody. In oneembodiment, the antibodies of the invention are supplied as a drysterilized lyophilized powder or water free concentrate in ahermetically sealed container and can be reconstituted, e.g., with wateror saline to the appropriate concentration for administration to asubject. Preferably, the antibodies of the invention are supplied as adry sterile lyophilized powder in a hermetically sealed container at aunit dosage of at least 5 mg, more preferably at least 10 mg, at least15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg,or at least 75 mg. The lyophilized antibodies of the invention should bestored at between 2 and 8° C. in their original container and theantibodies should be administered within 12 hours, preferably within 6hours, within 5 hours, within 3 hours, or within 1 hour after beingreconstituted. In an alternative embodiment, antibodies of the inventionare supplied in liquid form in a hermetically sealed containerindicating the quantity and concentration of the antibody, fusionprotein, or conjugated molecule. Preferably, the liquid form of theantibodies are supplied in a hermetically sealed container at least 1mg/ml, more preferably at least 2.5 mg/ml, at least 5 mg/ml, at least 8mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least50 mg/ml, at least 100 mg/ml, at least 150 mg/ml, at least 200 mg/ml ofthe antibodies.

The amount of the composition of the invention which will be effectivein the treatment, prevention or amelioration of one or more symptomsassociated with a disorder can be determined by standard clinicaltechniques. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thecondition, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

For antibodies encompassed by the invention, the dosage administered toa patient is typically 0.0001 mg/kg to 100 mg/kg of the patient's bodyweight. Preferably, the dosage administered to a patient is between0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kgor 0.01 to 0.10 mg/kg of the patient's body weight. Generally, humanantibodies have a longer half-life within the human body than antibodiesfrom other species due to the immune response to the foreignpolypeptides. Thus, lower dosages of human antibodies and less frequentadministration is often possible. Further, the dosage and frequency ofadministration of antibodies of the invention or fragments thereof maybe reduced by enhancing uptake and tissue penetration of the antibodiesby modifications such as, for example, lipidation.

In one embodiment, the dosage of the antibodies of the inventionadministered to a patient are 0.01 mg to 1000 mg/day, when used assingle agent therapy. In another embodiment the antibodies of theinvention are used in combination with other therapeutic compositionsand the dosage administered to a patient are lower than when saidantibodies are used as a single agent therapy.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion, by injection, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. Preferably,when administering an antibody of the invention, care must be taken touse materials to which the antibody or the fusion protein does notabsorb.

In another embodiment, the compositions can be delivered in a vesicle,in particular a liposome (See Langer, Science 249:1527-1533 (1990);Treat et al., in Liposomes in the Therapy of Infectious Disease andCancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365(1989); Lopez-Berestein, ibid., pp. 3 17-327; see generally ibid.) (eachof which is incorporated by reference in its entirety).

In yet another embodiment, the compositions can be delivered in acontrolled release or sustained release system. Any technique known toone of skill in the art can be used to produce sustained releaseformulations comprising one or more antibodies of the invention. See,e.g., U.S. Pat. No. 4,526,938; PCT publication WO 91/05548; PCTpublication WO 96/20698; Ning et al., 1996, “IntratumoralRadioimmunotheraphy of a Human Colon Cancer Xenograft Using aSustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Song et al.,1995, “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,”PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek etal., 1997, “Biodegradable Polymeric Carriers for a bFGF Antibody forCardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact.Mater. 24:853-854; and Lam et al., 1997, “Microencapsulation ofRecombinant Humanized Monoclonal Antibody for Local Delivery,” Proc.Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which isincorporated herein by reference in its entirety. In one embodiment, apump may be used in a controlled release system (See Langer, supra;Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:20; Buchwald et al., 1980,Surgery 88:507; and Saudek et al., 1989, N Engl. J. Med. 321:574). Inanother embodiment, polymeric materials can be used to achievecontrolled release of antibodies (see e.g., Medical Applications ofControlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.(1974); Controlled Drug Bioavailability, Drug Product Design andPerformance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger andPeppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; See alsoLevy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol.25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No.5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat.No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154;and PCT Publication No. WO 99/20253). Examples of polymers used insustained release formulations include, but are not limited to,poly(-hydroxy ethyl methacrylate), poly(methyl methacrylate),poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylicacid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides(PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In yetanother embodiment, a controlled release system can be placed inproximity of the therapeutic target (e.g., the lungs), thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).In another embodiment, polymeric compositions useful as controlledrelease implants are used according to Dunn et al. (See U.S. Pat. No.5,945,155). This particular method is based upon the therapeutic effectof the in situ controlled release of the bioactive material from thepolymer system. The implantation can generally occur anywhere within thebody of the patient in need of therapeutic treatment. In anotherembodiment, a non-polymeric sustained delivery system is used, whereby anon-polymeric implant in the body of the subject is used as a drugdelivery system. Upon implantation in the body, the organic solvent ofthe implant will dissipate, disperse, or leach from the composition intosurrounding tissue fluid, and the non-polymeric material will graduallycoagulate or precipitate to form a solid, microporous matrix (See U.S.Pat. No. 5,888,533). Each of the foregoing references is incorporatedherein in its entirety.

Controlled release systems are discussed in the review by Langer (1990,Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore therapeutic agents of the invention. See, e.g., U.S. Pat. No.4,526,938; International Publication Nos. WO 91/05548 and WO 96/20698;Ning et al., 1996, Radiotherapy & Oncology 39:179-189; Song et al.,1995, PDA Journal of Pharmaceutical Science & Technology 50:372-397;Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater.24:853-854; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel.Bioact. Mater. 24:759-760, each of which is incorporated herein byreference in its entirety.

In a specific embodiment where the composition of the invention is anucleic acid encoding an antibody, the nucleic acid can be administeredin vivo to promote expression of its encoded antibody, by constructingit as part of an appropriate nucleic acid expression vector andadministering it so that it becomes intracellular, e.g., by use of aretroviral vector (See U.S. Pat. No. 4,980,286), or by direct injection,or by use of microparticle bombardment (e.g., a gene gun; Biolistic,Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (See e.g.,Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc.Alternatively, a nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression by homologousrecombination.

For antibodies, the therapeutically or prophylactically effective dosageadministered to a subject is typically 0.1 mg/kg to 200 mg/kg of thesubject's body weight. Preferably, the dosage administered to a subjectis between 0.1 mg/kg and 20 mg/kg of the subject's body weight and morepreferably the dosage administered to a subject is between 1 mg/kg to 10mg/kg of the subject's body weight. The dosage and frequency ofadministration of antibodies of the invention may be reduced also byenhancing uptake and tissue penetration (e.g., into the lung) of theantibodies or fusion proteins by modifications such as, for example,lipidation.

Treatment of a subject with a therapeutically or prophylacticallyeffective amount of antibodies of the invention can include a singletreatment or, preferably, can include a series of treatments. In apreferred example, a subject is treated with antibodies of the inventionin the range of between about 0.1 to 30 mg/kg body weight, one time perweek for between about 1 to 10 weeks, preferably between 2 to 8 weeks,more preferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. In other embodiments, the pharmaceuticalcompositions of the invention are administered once a day, twice a day,or three times a day. In other embodiments, the pharmaceuticalcompositions are administered once a week, twice a week, once every twoweeks, once a month, once every six weeks, once every two months, twicea year or once per year. It will also be appreciated that the effectivedosage of the antibodies used for treatment may increase or decreaseover the course of a particular treatment.

6.4.1 Pharmaceutical Compositions

The compositions of the invention include bulk drug compositions usefulin the manufacture of pharmaceutical compositions (e.g., impure ornon-sterile compositions) and pharmaceutical compositions (i.e.,compositions that are suitable for administration to a subject orpatient) which can be used in the preparation of unit dosage forms. Suchcompositions comprise a prophylactically or therapeutically effectiveamount of a prophylactic and/or therapeutic agent disclosed herein or acombination of those agents and a pharmaceutically acceptable carrier.Preferably, compositions of the invention comprise a prophylactically ortherapeutically effective amount of humanized antibodies of theinvention and a pharmaceutically acceptable carrier.

In one particular embodiment, the pharmaceutical composition comprisesof a therapeutically effective amount of a humanized antibody or afragment thereof that binds FcγRIIB with a greater affinity than saidantibody or a fragment thereof binds FcγRIIA, a cytotoxic antibody thatspecifically binds a cancer antigen, and a pharmaceutically acceptablecarrier. In another embodiment, said pharmaceutical composition furthercomprises one or more anti-cancer agents.

In another particular embodiment, the pharmaceutical compositioncomprises (i) a therapeutically effective amount of a humanized antibodyor fragment thereof that specifically binds the extracellular domain ofhuman FcγRIIB and blocks the Fc binding site of human FcγRIIB; (ii) acytotoxic antibody that specifically binds a cancer antigen; and (iii) apharmaceutically acceptable carrier.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant(complete and incomplete), excipient, or vehicle with which thetherapeutic is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like.

Generally, the ingredients of compositions of the invention are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include, but are not limitedto, those formed with anions such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withcations such as those derived from sodium, potassium, ammonium, calcium,ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The present invention also provides pharmaceutical compositions and kitscomprising a FcγRIIB antagonist for use in the prevention, treatment,management, or amelioration of a B-cell malignancy, or one or moresymptoms thereof. In particular, the present invention providespharmaceutical compositions and kits comprising a humanized FcγRIIBantibody or an antigen-binding fragment thereof

6.4.2 Kits

The invention provides a pharmaceutical pack or kit comprising one ormore containers filled with humanized antibodies of the invention.Additionally, one or more other prophylactic or therapeutic agentsuseful for the treatment of a disease can also be included in thepharmaceutical pack or kit. The invention also provides a pharmaceuticalpack or kit comprising one or more containers filled with one or more ofthe ingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises one or more humanizedantibodies of the invention. In another embodiment, a kit furthercomprises one or more other prophylactic or therapeutic agents usefulfor the treatment of cancer, in one or more containers. In anotherembodiment, a kit further comprises one or more cytotoxic antibodiesthat bind one or more cancer antigens associated with cancer. In certainembodiments, the other prophylactic or therapeutic agent is achemotherapeutic. In other embodiments, the prophylactic or therapeuticagent is a biological or hormonal therapeutic.

6.5 Characterization and Demonstration of Therapeutic Utility

Several aspects of the pharmaceutical compositions or prophylactic ortherapeutic agents of the invention are preferably tested in vitro,e.g., in a cell culture system, and then in vivo, e.g., in an animalmodel organism, such as a rodent animal model system, for the desiredtherapeutic activity prior to use in humans. For example, assays whichcan be used to determine whether administration of a specificpharmaceutical composition is indicated, include cell culture assays inwhich a patient tissue sample is grown in culture, and exposed to orotherwise contacted with a pharmaceutical composition, and the effect ofsuch composition upon the tissue sample is observed, e.g., inhibition ofor decrease in growth and/or colony formation in soft agar or tubularnetwork formation in three-dimensional basement membrane orextracellular matrix preparation. The tissue sample can be obtained bybiopsy from the patient. This test allows the identification of thetherapeutically most effective prophylactic or therapeutic molecule(s)for each individual patient. Alternatively, instead of culturing cellsfrom a patient, therapeutic agents and methods may be screened usingcells of a tumor or malignant cell line. In various specificembodiments, in vitro assays can be carried out with representativecells of cell types involved in an autoimmune or inflammatory disorder(e.g., T cells), to determine if a pharmaceutical composition of theinvention has a desired effect upon such cell types. Many assaysstandard in the art can be used to assess such survival and/or growth;for example, cell proliferation can be assayed by measuring ³H-thymidineincorporation, by direct cell count, by detecting changes intranscriptional activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell cycle markers; cell viability can be assessed bytrypan blue staining, differentiation can be assessed visually based onchanges in morphology, decreased growth and/or colony formation in softagar or tubular network formation in three-dimensional basement membraneor extracellular matrix preparation, etc. Additional assays include raftassociation, CDC, ADCC and apoptosis assays as known in the art.

Combinations of prophylactic and/or therapeutic agents can be tested insuitable animal model systems prior to use in humans. Such animal modelsystems include, but are not limited to, rats, mice, chicken, cows,monkeys, pigs, dogs, rabbits, etc. Any animal system well-known in theart may be used. In a specific embodiment of the invention, combinationsof prophylactic and/or therapeutic agents are tested in a mouse modelsystem. Such model systems are widely used and well-known to the skilledartisan. Prophylactic and/or therapeutic agents can be administeredrepeatedly. Several aspects of the procedure may vary such as thetemporal regime of administering the prophylactic and/or therapeuticagents, and whether such agents are administered separately or as anadmixture.

Preferred animal models for use in the methods of the invention are, forexample, transgenic mice expressing FcγR on mouse effector cells, e.g.,any mouse model described in U.S. Pat. No. 5,877,396 (which isincorporated herein by reference in its entirety). Transgenic mice foruse in the methods of the invention include, but are not limited to,mice carrying human FcγRIIIA, mice carrying human FcγRIIA, mice carryinghuman FcγRIIB and human FcγRIIIA, mice carrying human FcγRIIB and humanFcγRIIA.

Once the prophylactic and/or therapeutic agents of the invention havebeen tested in an animal model they can be tested in clinical trials toestablish their efficacy. Establishing clinical trials will be done inaccordance with common methodologies known to one skilled in the art,and the optimal dosages and routes of administration as well as toxicityprofiles of the compositions of the invention can be established usingroutine experimentation.

The anti-inflammatory activity of the combination therapies of inventioncan be determined by using various experimental animal models ofinflammatory arthritis known in the art and described in Crofford L. J.and Wilder R. L., “Arthritis and Autoimmunity in Animals”, in Arthritisand Allied Conditions: A Textbook of Rheumatology, McCarty et al.(eds.), Chapter 30 (Lee and Febiger, 1993). Experimental and spontaneousanimal models of inflammatory arthritis and autoimmune rheumaticdiseases can also be used to assess the anti-inflammatory activity ofthe combination therapies of invention. The following are some assaysprovided as examples, and not by limitation.

The principle animal models for arthritis or inflammatory disease knownin the art and widely used include: adjuvant-induced arthritis ratmodels, collagen-induced arthritis rat and mouse models andantigen-induced arthritis rat, rabbit and hamster models, all describedin Crofford L. J. and Wilder R. L., “Arthritis and Autoimmunity inAnimals”, in Arthritis and Allied Conditions: A Textbook ofRheumatology, McCarty et al. (eds.), Chapter 30 (Lee and Febiger, 1993),incorporated herein by reference in its entirety.

The anti-inflammatory activity of the combination therapies of inventioncan be assessed using a carrageenan-induced arthritis rat model.Carrageenan-induced arthritis has also been used in rabbit, dog and pigin studies of chronic arthritis or inflammation. Quantitativehistomorphometric assessment is used to determine therapeutic efficacy.The methods for using such a carrageenan-induced arthritis model isdescribed in Hansra P. et al., “Carrageenan-Induced Arthritis in theRat,” Inflammation, 24(2): 141-155, (2000;) incorporated by reference inits entirety. Also commonly used are zymosan-induced inflammation animalmodels as known and described in the art. A collagen-induced arthritis(CIA) is an animal model for the human autoimmune disease rheumatoidarthritis (RA) (Trenthom et al., 1977, J. Exp. Med. 146:857, which ishereby incorporated by reference herein in its entirety). This diseasecan be induced in many species by the administration of heterologoustype II collagen (Courtenay et al., 1980, Nature 283:665; and Cathcartet at, 1986, Lab. Invest. 54:26); with respect to animal models ofarthritis see, in addition, e.g., Holmdahl, R., 1999, Curr. Biol.15:R528-530 (each of these references is hereby incorporated byreference herein in its entirety).

The anti-inflammatory activity of the combination therapies of inventioncan also be assessed by measuring the inhibition of carrageenan-inducedpaw edema in the rat, using a modification of the method described inWinter C. A. et al., “Carrageenan-Induced Edema in Hind Paw of the Ratas an Assay for Anti-inflammatory Drugs” Proc. Soc. Exp. Biol Med. 111,544-547, (1962); incorporated by reference in its entirety. This assayhas been used as a primary in vivo screen for the anti-inflammatoryactivity of most NSAIDs, and is considered predictive of human efficacy.The anti-inflammatory activity of the test prophylactic or therapeuticagents is expressed as the percent inhibition of the increase in hindpaw weight of the test group relative to the vehicle dosed controlgroup.

Additionally, animal models for inflammatory bowel disease can also beused to assess the efficacy of the combination therapies of invention(Kim et al., 1992, Scand. J. Gastroentrol. 27:529-537; Strober, 1985,Dig. Dis. Sci. 30(12 Suppl):3S-10S (each of which is incorporated byreference in its entirety)). Ulcerative cholitis and Crohn's disease arehuman inflammatory bowel diseases that can be induced in animals.Sulfated polysaccharides including, but not limited to, amylopectin,carrageen, amylopectin sulfate, and dextran sulfate or chemicalirritants including, but not limited to, trinitrobenzenesulphonic acid(TNBS) and acetic acid can be administered to animals orally to induceinflammatory bowel diseases.

Animal models for asthma can also be used to assess the efficacy of thecombination therapies of invention. An example of one such model is themurine adoptive transfer model in which aeroallergen provocation of TH1or TH2 recipient mice results in TH effector cell migration to theairways and is associated with an intense neutrophilic (TH1) andeosinophilic (TH2) lung mucosal inflammatory response (Cohn et al.,1997, J. Exp. Med. 1861737-1747; incorporated by reference in itsentirety).

Animal models for autoimmune disorders can also be used to assess theefficacy of the combination therapies of invention. Animal models forautoimmune disorders such as type 1 diabetes, thyroid autoimmunity,systemic lupus eruthematosus, and glomerulonephritis have been developed(Flanders et al., 1999, Autoimmunity 29:235-246; Krogh et al., 1999,Biochimie 81:511-515; Foster, 1999, Semin. Nephrol. 19:12-24 (each ofwhich is incorporated by reference in its entirety)).

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for autoimmune and/orinflammatory diseases.

Toxicity and efficacy of the prophylactic and/or therapeutic protocolsof the instant invention can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Prophylacticand/or therapeutic agents that exhibit large therapeutic indices arepreferred. While prophylactic and/or therapeutic agents that exhibittoxic side effects may be used, care should be taken to design adelivery system that targets such agents to the site of affected tissuein order to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the prophylactic and/ortherapeutic agents for use in humans. The dosage of such agents liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any agent used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

The anti-cancer activity of the therapies used in accordance with thepresent invention also can be determined by using various experimentalanimal models for the study of cancer such as the SCID mouse model ortransgenic mice or nude mice with human xenografts, animal models, suchas hamsters, rabbits, etc. known in the art and described in Relevanceof Tumor Models for Anticancer Drug Development (1999, eds. Fiebig andBurger); Contributions to Oncology (1999, Karger); The Nude Mouse inOncology Research (1991, eds. Boven and Winograd); and Anticancer DrugDevelopment Guide (1997 ed. Teicher), herein incorporated by referencein their entireties.

The protocols and compositions of the invention are preferably tested invitro, and then in vivo, for the desired therapeutic or prophylacticactivity, prior to use in humans. Therapeutic agents and methods may bescreened using cells of a tumor or malignant cell line. Many assaysstandard in the art can be used to assess such survival and/or growth;for example, cell proliferation can be assayed by measuring ³H-thymidineincorporation, by direct cell count, by detecting changes intranscriptional activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell cycle markers; cell viability can be assessed bytrypan blue staining, differentiation can be assessed visually based onchanges in morphology, decreased growth and/or colony formation in softagar or tubular network formation in three-dimensional basement membraneor extracellular matrix preparation, etc.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to inrats, mice, chicken, cows, monkeys, rabbits, hamsters, etc., forexample, the animal models described above. The compounds can then beused in the appropriate clinical trials.

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for treatment or prevention ofcancer, inflammatory disorder, or autoimmune disease.

6.6 Diagnostic Methods

Labeled antibodies of the invention can be used for diagnostic purposesto detect, diagnose, or monitor diseases, disorders or infections. Theinvention provides for the detection or diagnosis of a disease, disorderor infection, particularly an autoimmune disease comprising: (a)assaying the expression of FcγRIIB in cells or a tissue sample of asubject using one or more antibodies that immunospecifically bind toFcγRIIB; and (b) comparing the level of the antigen with a controllevel, e.g., levels in normal tissue samples, whereby an increase in theassayed level of antigen compared to the control level of the antigen isindicative of the disease, disorder or infection.

Antibodies of the invention can be used to assay FcγRIIB levels in abiological sample using classical immunohistological methods asdescribed herein or as known to those of skill in the art (e.g., seeJalkanen et al., 1985, J. Cell. Biol. 101:976-985; Jalkanen et al.,1987, J. Cell. Biol. 105:3087-3096 (each of which is incorporated byreference in its entirety)). Other antibody-based methods useful fordetecting protein gene expression include immunoassays, such as theenzyme linked immunosorbent assay (ELISA) and the radioimmunoassay(RIA). Suitable antibody assay labels are known in the art and includeenzyme labels, such as, alkaline phosphatase, glucose oxidase;radioisotopes, such as iodine (¹²⁵I, ¹³¹I) carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹²¹In) and technetium (^(99m)Tc); luminescentlabels, such as luminol; and fluorescent labels, such as fluorescein andrhodamine.

One aspect of the invention is the detection and diagnosis of a disease,disorder, or infection in a human. In one embodiment, diagnosiscomprises: a) administering (for example, parenterally, subcutaneously,or intraperitoneally) to a subject an effective amount of a labeledantibody that immunospecifically binds to FcγRIIB; b) waiting for a timeinterval following the administration for permitting the labeledantibody to preferentially concentrate at sites in the subject whereFcγRIIB is expressed (and for unbound labeled molecule to be cleared tobackground level); c) determining background level; and d) detecting thelabeled antibody in the subject, such that detection of labeled antibodyabove the background level indicates that the subject has the disease,disorder, or infection. In accordance with this embodiment, the antibodyis labeled with an imaging moiety which is detectable using an imagingsystem known to one of skill in the art. Background level can bedetermined by various methods including, comparing the amount of labeledmolecule detected to a standard value previously determined for aparticular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ^(99m)Tc. The labeled antibodywill then preferentially accumulate at the location of cells whichcontain the specific protein. In vivo tumor imaging is described in S.W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodiesand Their Fragments.” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982); incorporated by reference in its entirety.

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In one embodiment, monitoring of a disease, disorder or infection iscarried out by repeating the method for diagnosing the disease, disorderor infection, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the subject usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patient using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

7. EXAMPLES

7.1 Humanization of Mouse Anti-CD32B MAB 2B6

RNA was converted to cDNA and the VH and VL segments were PCR amplifiedusing the RLM-RACE kit (Ambion, Inc.). Gene specific primers for the VHwere SJ15R, SEQ ID NO. 47 (5′ GGT CAC TGT CAC TGG CTC AGG G 3′) andSJ16R, SEQ ID NO. 48 (5′ AGG CGG ATC CAG GGG CCA GTG GAT AGA C3′). Genespecific primers for the VL were SJ17R, SEQ ID NO. 49 (5′GCA CAC GAC TGAGGC ACC TCC AGA TG 3′) and SJ18R, SEQ ID NO. 50 (5′ CGG CGG ATC CGA TGGATA CAG TTG GTG CAG CAT C3′). The RACE product was inserted into theplasmid pCR2.1-TOPO using a TOPO TA Cloning kit (Invitrogen, Inc.). Theresulting plasmids were then subjected to DNA sequencing to determinethe VH and VL sequences for 2B6. The resulting sequences were thentranslated and the predicted amino acid sequence determined for each.From these sequences, the framework (FR) and complementarity determining(CDR) regions were identified as defined by Kabat. The mouse VH was thenjoined to a human C-Gammal constant region and an Ig leader sequence andinserted into pCI-neo for mammalian expression. The mouse VL was joinedto a human C-kappa segment and an Ig leader sequence and also clonedinto pCI-neo for mammalian expression.

The humanized 2B6 VH consists of the FR segments from the human germlineVH segment VH1-18 and JH6, and the CDR regions of the 2B6 VH. Thehumanized 2B6 VL consists of the FR segments of the human germline VLsegment VK-A26 and JK4, and the CDR regions of 2B6 VL. The humanized VHand VL segments were assembled de novo from oligonucleotides combinedand amplified by PCR. The resulting fragment was then combined by PCRwith a leader sequence and the appropriate constant region segmentcloned into the expression vector pCI-neo as a Nhe I-EcoR I fragment.The DNA sequence of the resulting plasmids was confirmed by sequenceanalysis. For the VL, none of the plasmids analyzed had a perfectlycorrect sequence. The two best inserts were combined to reduce thenumber of incorrect positions, then these positions were corrected bysite-directed mutagenesis. After this procedure light chain segmentshaving predicted humanized 2B6 VL sequence were identified.

The alignment of the amino acid sequences of mouse 2B6 VH, humanized 2B6VH1, VH1-18 and human JH6 is shown in FIG. 1A. FIG. 1B shows thealignment of amino acid sequences of murine 2B6VL, human 2B6VL-1, human2B6VL-2; human 2B6VL-3, and human JK4. The first amino acid in thehumanized 2B6VL CDR2 was found to be asparagine (see, e.g., amino acidnumber 1 of SEQ ID NO:9), presumably allowing N-linked glycosylation andpossibly affecting binding of the antibody. For this reason amino acidnumber 1 of the light chain variable domain CDR2 was substituted withtyrosine (h2B6 VL-2, h2B6 VL-3) or glutamic acid (h2B6 VL-5; amino acid50 of SEQ ID NO:62) to remove the glycosylation site (FIG. 2B). h2B6VL-5 (SEQ ID NO:62) additionally contains a substitution withphenylalanine at amino acid number 21 of framework region 1 (amino acidnumber 21 of SEQ ID NO:62), which corresponds to the amino acid at thesame position in the donor antibody light chain variable domain. FIG. 2Ashows the alignment of the heavy chain variable regions, h2B6 VH-1 andh2B6 VH-3 (SEQ ID NO:68). h2B6 VH-3 contains a substitution at aminoacid number 13 of framework region 2 (amino acid number 48 of SEQ IDNO:68), and substitutions with valine at amino acid number 6 offramework region 3 (amino acid 72 of SEQ ID NO:68), which corresponds tothe amino acid at the same position in the donor antibody heavy chainvariable domain.

7.2 Expression and Characterization of the Humanized 2B6 Heavy and LightChains.

Experiment 1:

The hu2B6 heavy chain (HC) expression plasmid was co-transfectedtogether with ch2B6 light chain (LC) into HEK-293 cells. At the sametime, the ch2B6HC was co-transfected with the ch2B6LC. After three daysin culture the amount of human IgG expressed was quantitated by ELISA.Binding to dimeric soluble FcγRIIb-Fc was then determined by ELISAassay.

Protocol for ELISA Assay:

2.5 ng/well of soluble FcγRIIb-Fc was captured on 96-well Maxisorpplates by mouse anti-FcγRIIB antibody 3H7 at room temperature for 1hour. A serial of two-fold dilution of conditioned medium of ch2B6,h2B6Hc/Ch2B6Lc, h2B6 v1.1 (comprising the heavy chain variable domainh2B6 VH-1 (FIG. 1A and FIG. 2A) and light chain variable domain h2B6VL-1 (FIG. 1-B and FIG. 2B)) or h2B6 v3.5 (comprising the heavy chainvariable domain h2B6 VH-3 (FIG. 2A; SEQ ID NO:68) and light chainvariable domain h2B6 VL-5 (FIG. 2B; SEQ ID NO:62)) starting from 25ng/well was added to the each well. The plate was incubated at roomtemperature for 1 hour, then binding was detected by HRP conjugatedF(ab′)₂ goat anti human IgG F(ab)′₂ specific secondary antibody. Afterincubation with the secondary antibody for approximately 45 minutes, theplate was developed using a TMB substrate. After 5 minutes incubation,the reaction was stopped by 1% H₂SO₄. The OD₄₅₀ nm was read by SOFTmaxprogram. Between each step, the plates were washed 3 times with PBS/0.1%Tween20. Plates were blocked by 0.5% BSA in PBS/0.1% Tween 20 for 30mins at room temperature before adding soluble FcγRIIb-Fc.

Results:

The results of the ELISA assay with hu2B6HC/ch2B6LC and ch2B6(ch2B6HC/ch2B6LC) are depicted in FIG. 3, which indicate that thehu2B6HC/ch2B6LC mAb bound to the receptor with similar affinity as thech2B6HC/ch2B6LC mAb. A second ELISA was performed as for ch2B6, h2B61.1, and h2B6 3.5 as described with the exception that solubleFcγRIIB-Fc was captured directly on the 96-well Maxisorp plates at 4° C.over night (FIG. 4). The results indicate that h2B6 3.5 bound to thereceptor with similar affinity to ch2B6, and substantially greateraffinity than ch2B6 1.1, which comprises the glycosylation site in CDRof the light chain variable domain.

FACS analysis was then performed to measure the binding of the mAbs toDaudi cells.

Protocol for FACS Analysis.

Approximately 10⁶ Daudi cells were used for each antibody staining Cellswere washed once with PBS. Primary antibodies (Ch2B6, Hu2B6Hc/ch2B6Lc,human IgG1) were diluted into 0.5, 0.1, 0.02 μg/mL in PBS/1% BSA and 100μL of diluted antibodies was transferred to the cells. After 30 minsincubation at 4° C., cells were washed once with 1 mL PBS/1% BSA. PEconjugated F(ab′)₂ fragment of goat anti human IgG Fc specific (JacksonImmunoReseach, Inc.) was used as secondary antibody at 1:1000 dilution.After 30 mins incubation at 4° C., cells were washed once with 1 mLPBS/1% BSA. The cells were then resuspended in 500 μL of PBS/1% BSA andsubjected to be FACS analysis.

Results:

The results indicate that hu2B6HC/ch2B6LC mAb binds to this human B celltumor line with the same affinity as the chimeric mAb (Table 6).

TABLE 6 Primary Antibody Concentration (μg/ml) Mean Fluorescence HumanIgG1 0.5 9.49 0.1 N/A 0.02 N/A Ch2B6 0.5 647.48 0.1 511.85 0.02 172.68Hu2B6Hc/Ch2B6Lc 0.5 648.99 0.1 546.46 0.02 196.93

Experiment 2:

Transfections of HEK-293 cells were performed using the followingcombinations: hu2B6HC/hu2B6LC, hu2B6HC/ch2B6LC, ch2B6HC/hu2B6LC andch2B6HC/ch2B6LC. After three days in culture the amount of human IgGexpressed was quantitated by ELISA using the protocol described above.Binding to dimeric soluble FcγRIIb-Fc was then determined by ELISA. Theresults of this experiment, depicted in FIG. 5, indicated that all ofthe mAbs bound to the receptor with similar affinity. FACS analysis wasthen performed using the protocol described above to measure the bindingof the mAbs to Daudi cells (Table 7). The results indicate thathu2B6HC/hu2B6LC mAb binds to this human B cell tumor line with the sameaffinity as the ch2B6 mAb.

TABLE 7 Primary Antibody Concentration (ug/ml) Mean Fluorescence HumanIgG1 0.5 6.07 0.1 N/A 0.02 N/A Ch2B6 0.5 551.52 0.1 514.69 0.02 168.17Hu2B6 0.5 628.82 0.1 618.13 0.02 228.74

7.3 Generation, Expression and Binding of HU2B6LC Variants.

There is a consensus sequence of N-glycosylation site (Asn-Xaa-Ser/Thr)in the Hu2B6LC CDR2 region (Asn₅₀-Val-Ser). To eliminate theglycosylation at residue 50 and thus limit potential variation inproduction as well as potential immunogenicity in a pharmaceuticalapplication, other amino acids were substituted at the position 50 usingsite-directed mutagenesis (Stratagene kit). Two different versions ofHu2B6LC were generated, Hu2B6LC-N50Y Hu2B6LC-N50Y,V51A. These aminoacids were chosen because Tyrosine is the human acceptor residue atCDRL2 position 50 and Alanine is the residue at CDRL2 position 51 in thehuman germline gene segment.

Transfections of HEK-293 cells were performed using the followingcombinations: hu2B6HC/hu2B6LC; hu2B6HC/hu2B6LC(N50Y);hu2B6HC/hu2B6LC(N50Y,V51A); ch2B6HC/ch2B6LC. After three days in culturethe amount of human IgG expressed was quantitated by an ELISA assay,using the method described above. Binding to dimeric soluble FcγRIIb-Fcwas determined by ELISA assay. The results of this experiment, depictedin FIG. 6, indicated that all of the mAbs bound to the receptor withsimilar affinity. FACS analysis was then performed to measure thebinding of the mAbs to Daudi cells (Table 8). The results demonstratethat the two variants of hu2B6LC/hu2B6HC mAbs bind to this human B celltumor line with the same affinity as the ch2B6 mAb.

TABLE 8 Primary Antibody Concentration (μg/ml) Mean Fluorescence HumanIgG1 0.5 1.23 0.1 N/A 0.02 N/A Ch2B6 0.5 192.88 0.1 141.01 0.02 45.59Hu2B6 0.5 201.69 0.1 174.37 0.02 58.65 Hu2B6 N50Y 0.5 191.16 0.1 134.560.02 40.14 Hu2B6N50Y, V51A 0.5 167.16 0.1 133.83 0.02 45.95

7.4 Binding of MAbs TO FcRIIA

Protocol for ELISA assay: 100 ng/well of soluble FcγIIA in carbonatebuffer was coated on 96-well Maxisorp plates at 4° C. overnight. Aserial of two-fold dilution of conditioned medium of Ch2B6;hu2B6HC/hu2B6LC; hu2B6HC/hu2B6LC(N50Y); hu2B6HC/hu2B6LC(N50Y,V51A); andpurified IV.3 starting from 25 ng/well was added to the each well. Theplate was incubated at room temperature for 1 hour. The binding wasdetected by HRP conjugated F(ab′)2 goat anti human IgG F(ab′)2 specificsecondary antibody for Ch2B6 and all hu2B6 samples and HRP conjugatedF(ab′)2 goat anti mouse IgG (H+L) secondary antibody for IV.3. Afterincubation with the secondary antibody for approximately 45 minutes, theplate was developed using a TMB substrate. After 5 mins incubation, thereaction was stopped by 1% H₂SO₄. The OD₄₅₀ nm was ready by SOFTmaxprogram. Between each step, the plates were washed 3 times with PBS/0.1%Tween 20. The plates were blocked by 0.5% BSA in PBS/0.1% Tween 20 for30 mins at room temperature before adding the serial diluted antibodies.

Results:

These data show that the humanized 2B6 antibody did not lose its abilityto selectively bind CD32B during the humanization process (FIG. 7). Insummary IV.3 (a murine Mab against FcγIIA) binds FcγIIA while chimericand humanized 2B6 does not.

7.5 Fc Mutant Mediated Tumor Growth Control in an In Vivo Tumor Model

Fc mutations previously identified as conferring enhanced affinity forFcγIIIA and/or FcγIIA to FcγRIIB antibodies were further analyzed forrelative efficacy of tumor control using an in vivo tumor model system(see, U.S. Provisional Application 60/707,419 filed Aug. 10, 2005;hereby incorporated by reference in its entirety)).

Materials and Methods

Antibodies harboring Fc mutants were tested for anti-tumor activity in amurine xenograft system. Balbc/nude mice were subcutaneously injectedwith 5×10⁶ Daudi cells and subsequently monitored for general signs ofillness, e.g. weight gain/loss and grooming activity. Without treatment,this model system results in 100% mortality with an average survivaltime of approximately 2 weeks post tumor cell inoculation. Treatmentconsists of doses of wild-type antibody or antibody comprising a variantFc region administered at weekly intervals. Animals administered bufferalone at the same intervals served as a control. Tumor weight wascalculated based on the estimated volume of the subcutaneous tumoraccording to the formula (width²×length)/2.

Results

At weekly intervals, mice inoculated with Daudi cells received wild-typehumanized 2B6 (“h2B6”), humanized 2B6 comprising an Fc region having243L, 292P, 300L, 3051, and 396L or buffer alone. Wild-type and Fcmutant h2B6 antibody showed similar levels of tumor suppression at thehighest dose schedule tested, weekly doses of 25 mg (FIGS. 7 A and B).However, significant differences in antibody efficacy were observed whendosages were reduced. 100 and 10 fold reduction in wild-type h2B6dosages provided no greater tumor control than administration of bufferalone (FIG. 8A). In contrast, the Fc mutant h2B6 provided significantprotection at weekly doses of 2.5 μg and at least limited protection atweekly doses of 0.25 μg (FIG. 8B).

The protection conferred by even the lowest dose of Fc mutant antibodywas confirmed in survival comparisons. At 11 weeks, 4 out of 7 miceremained alive in the group treated with 0.25 ng doses of Fc mutant h2B6compared to only 1 out of 7 in the group treated with the same dose ofwild-type h2B6 (FIGS. 9A and 9B).

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

Various references are cited herein, the disclosure of which areincorporated by reference in their entirety.

1. A method of treating cancer in a patient having a cancercharacterized by a cancer antigen, said method comprising administeringto said patient a therapeutically effective amount of: (A) a firstantibody that comprises an antigen-binding domain that specificallybinds the extracellular domain of human FcγRIIB via said antigen-bindingdomain, wherein said first antibody comprises a heavy chain variabledomain comprising the amino acid sequence of SEQ ID NO:37 and a lightchain variable domain comprising the amino acid sequence of SEQ IDNO:46, or a fragment of said first antibody that comprises saidantigen-binding domain and specifically binds said extracellular domainof human FcγRIIB via said antigen-binding domain; and (B) a secondantibody that specifically binds said cancer antigen and is cytotoxic.2. The method of claim 1, wherein said first antibody is a humanizedantibody that comprises a human Fc domain, and wherein said human Fcdomain of said humanized antibody comprises at least one amino acidmodification.
 3. The humanized antibody of claim 2, wherein said atleast one amino acid modification comprises a substitution at position243, a substitution at position 292, a substitution at position 300, asubstitution at position 305, and a substitution at position 396 andwherein said positions are according to Kabat.
 4. The humanized antibodyof claim 3, wherein the substitution at position 243 is a substitutionwith leucine, the substitution at position 292 is a substitution withproline, the substitution at position 300 is a substitution withleucine, the substitution at position 305 is a substitution withisoleucine, and the substitution at position 396 is a substitution withleucine and wherein said positions are according to Kabat.
 5. The methodof claim 2, wherein said at least one amino acid modification comprisesa substitution at position 265 or 297, wherein said positions areaccording to Kabat.
 6. The method of claim 5, wherein said position 265is substituted with alanine, or said position 297 is substituted withglutamine.
 7. The method of claim 1, wherein said fragment of said firstantibody is selected from the group consisting of an F(ab) fragment, anF(ab′) fragment, an F(ab′)₂ fragment, an Fv fragment, an scFv fragment,and an sdFv fragment.
 8. The method of claim 2, wherein said firstantibody is a single chain antibody.
 9. The method of claim 1, whereinsaid second antibody is operably linked to a heterologous polypeptide.10. The method of claim 1, wherein said second antibody is conjugated toa therapeutic agent.
 11. The method of claim 10, wherein saidtherapeutic agent is a cytotoxin.
 12. The method of claim 1, whereinsaid cancer antigen is a melanoma cancer antigen, a G-Antigen (GAGE),N-acetylglucosaminyltransferase, beta-catenin, MUM-1, CDK4, HER-2/neu,human papillomavirus-E6, human papillomavirus-E7, or MUC-1.
 13. Themethod of claim 1, wherein said cancer antigen is a breast, ovarian,stomach, uterine, prostate, cervical, or pancreatic carcinoma antigen.14. The method of claim 1, further comprising the administration of anadditional cancer therapy.
 15. The method of claim 14, wherein saidadditional cancer therapy is selected from the group consisting ofchemotherapy, immunotherapy, radiation therapy, hormonal therapy, orsurgery.
 16. The method of claim 1, wherein said patient is a human. 17.The method of claim 2, wherein said human Fc domain of said humanizedantibody exhibits increased affinity to an Fc activation receptor,relative to the affinity of a wild-type Fc domain.
 18. The method ofclaim 17, wherein said Fc activation receptor is FcγRIIIA.